CN112111001A

CN112111001A - Method for synthesizing thymosin T alpha-1

Info

Publication number: CN112111001A
Application number: CN201910530695.7A
Authority: CN
Inventors: 姚志军; 宓鹏程; 陶安进; 袁建成
Original assignee: Hybio Pharmaceutical Wuhan Co ltd; Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Wuhan Co ltd; Hybio Pharmaceutical Co Ltd
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2020-12-22
Anticipated expiration: 2039-06-19
Also published as: WO2020252883A1; CN112111001B

Abstract

The invention discloses a method for synthesizing thymosin T alpha-1, which comprises the following steps: 1) according to Fmoc solid phase synthesis method, coupling amino acids to a solid phase carrier one by one to synthesize 5 peptide resin with side chain belt protecting groups; 2) preparing 7 peptide resin with an Hmb protecting group; 3) continuously coupling residues at 1-21 positions according to an Fmoc solid phase synthesis method to obtain 28 peptide resin; 4) and (3) cracking the 28 peptide resin to remove the C-terminal resin and all protecting groups to obtain the crude T alpha-l peptide. According to the invention, the Hmb protecting group is introduced into the Val at the 23-position, so that beta folding in the peptide chain coupling process can be effectively reduced, the reaction efficiency is further improved, and only the Val at the 23-position is selected to introduce the Hmb protecting group, so that the production cost is saved, the effect of improving beta folding is very obvious, and the total yield and the purity of the product are improved; the HPLC purity of the crude peptide T alpha l prepared by the method can reach 98 percent, and only a small amount of impurities exist, so that compared with the prior art, the method greatly simplifies the difficulty of subsequent purification, saves the purification cost, and is beneficial to industrial application.

Description

Method for synthesizing thymosin T alpha-1

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to synthesis of nonacetylated thymalfasin.

Background

The thymus contains a plurality of hormones belonging to three types of alpha, beta and gamma, and induces the mature differentiation of T cells together. The main active component of the thymosin is thymosin alpha 1(T alpha 1) consisting of 28 amino acids, and the chemically synthesized commodity is N-terminal acetylated thymosin alpha 1(T alpha 1) -thymalfasin. Its molecular weight is 3108.28, molecular formula is C₁₂₉H₂₁₅N₃₃O₅₅The peptide sequence is N-Acetyl-Ser¹-Asp²-Ala³-Ala⁴-Val⁵-Asp⁶-Thr⁷-Ser⁸-Ser⁹-Glu¹⁰-Ile¹¹-Thr¹²-Thr¹³-Lys¹⁴-Asp¹⁵-Leu¹⁶-Lys¹⁷-Glu¹⁸-Lys¹⁹-Lys²⁰-Glu²¹-Val²²-Val²³-Glu²⁴-Glu²⁵-Ala²⁶-Glu²⁷-Asn²⁸-OH. T α l is often used clinically as an immunopotentiator or immunomodulator for the treatment of various immunodeficiency and immunosuppressive diseases.

At present, the synthesis method of thymalfasin mainly adopts Fmoc solid-phase synthesis. Since thymalfasin is a difficult polypeptide, beta sheets are formed during synthesis, resulting in difficulties in synthesis. In the beta-sheet structure, there are a large number of hydrogen bonds on both sides of the backbone. These hydrogen bonds can tightly group the peptide chains together. The solubility of the peptide is worse when the proportion of the beta-sheet structure is larger in the whole backbone. This feature can make the peptide reaction difficult. And the purity can only reach about 50% by adopting the traditional Fmoc solid phase sequential coupling method, and even if polypeptide fragment raw materials are adopted for coupling, such as patent CN201410333844, the purity is only 62-66%. Lower purity makes yield reduction and purification difficult, which in turn leads to increased production costs.

It was found that backbone protecting groups can disrupt the hydrogen bonding of the peptide chain, thereby disrupting the beta-sheet and improving the solubility of the peptide. However, the introduction of low-activity framework protecting groups such as Dmb and Hmb makes the coupling of a subsequent residue difficult, especially for amino acids with larger steric hindrance, such as valine Val, so that the site of the framework protecting group is limited. And the skeleton protecting groups such as Hmsb and Hnb with high activity involve complicated removal steps and various side reactions, so that the purity of the synthesized product is not high.

In view of the above situation, the present invention adopts Fmoc solid phase synthesis to introduce a skeleton N modification protecting group Hmb into Val at position 23, so that the synthesized 28-peptide crude peptide has an unobtainable purity of 98%.

Disclosure of Invention

In order to solve the problems of the background art, the present invention provides a method for synthesizing thymosin T alpha-1.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a method for synthesizing thymosin T alpha-1 comprises the following steps:

1) according to Fmoc solid phase synthesis method, amino acids are coupled to a solid phase carrier one by one, and 5 peptide resin of side chain belt protecting groups is synthesized: NH (NH)₂-glu (otbu) -Ala-glu (otbu) - β -asp (otbu) -amino resin;

2) preparation of 7-peptide resin with Hmb protecting group:

NH₂-Val- (Hmb) Val-glu (otbu) -Ala-glu (otbu) - β -asp (otbu) -amino resin;

3) continuously coupling residues at 1-21 positions according to an Fmoc solid phase synthesis method to obtain 28 peptide resin:

NH₂-ser (tbu) -Asp- (OtBu) -Ala-Val-Asp (OtBu) -thr (tbu) -ser (tbu) -glu (OtBu) -Ile-thr (tbu) -lys (boc) -Asp (OtBu) -Leu-lys (boc) -glu (OtBu) -Val- (Hmb) Val-glu (OtBu) -Ala-glu (OtBu) - β -Asp (OtBu) -amino resin;

4) and (3) cracking the 28 peptide resin to remove the C-terminal resin and all protecting groups to obtain the crude T alpha-l peptide.

Further, the preparation of the 5-peptide resin in 1) specifically comprises the following steps:

a. adding the solid phase carrier into a solid phase synthesis reactor, fully swelling with a solvent, removing the Fmoc protecting group with 20% piperidine/DMF solution, and washing;

b. adding Fmoc-Asp-OtBu and a coupling agent into DMF for reaction for a period of time, adding the mixture into a solid phase synthesis reactor, carrying out nitrogen blowing reaction for a period of time, and washing; then removing Fmoc protecting group by using 20% piperidine/DMF solution and washing; the above procedure was repeated to couple Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH and Fmoc-Glu (OtBu) -OH in this order.

Further, the 7-peptide resin with Hmb protecting group in 2) was prepared by the following method: fmoc- (Fmoc-Hmb) -Val-OH is used as a raw material to be introduced, the 5-position Val is coupled by a symmetrical anhydride method after the Fmoc protecting group is removed after the 5-position peptide resin obtained in the step 1) is coupled with the Fmoc- (Fmoc-Hmb) -Val-OH according to the coupling method the same as that in the step 1), and 7-position peptide resin is obtained.

Further, the 7-peptide resin with Hmb protecting group in 2) can be prepared by the following method: introducing a dipeptide fragment Fmoc-Val- (Fmoc-Hmb) -Val-OH serving as a raw material, coupling the Fmoc-Val- (Fmoc-Hmb) -Val-OH to the 5 peptide resin obtained in the step 1) according to the same coupling method as that in the step 1), and removing an Fmoc protecting group to obtain the 7 peptide resin.

Further, the 7-peptide resin with Hmb protecting group in 2) can be prepared by the following method: forming a framework protecting group in situ through reductive amination reaction, treating the 5-peptide resin obtained in the step 1) with p-methoxysalicylaldehyde, reducing with sodium borohydride or sodium cyanoborohydride, washing, and coupling with 22-Val to obtain the 7-peptide resin.

Further, the preparation of the 28 peptide resin in 3) specifically comprises the following steps:

a. adding the 7-peptide resin prepared in the step 2) into a solid phase synthesis reactor, fully swelling with a solvent, and then removing an Fmoc protecting group with 20% piperidine/DMF solution and washing;

b. adding Fmoc-Glu (OtBu) -OH and a coupling agent into DMF for reaction for a period of time, adding into a solid phase synthesis reactor, reacting with nitrogen for a period of time, and washing; then removing Fmoc protecting group by using 20% piperidine/DMF solution and washing; repeating the above process to couple Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH sequentially, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH.

Further, the solid phase carrier in 1) is selected from Rink Amide resin, Rink Amide-AM resin or Rink Amide-MBHA resin, and the substitution degree of the solid phase carrier is 0.1-0.7 mmol/g. The substitution degree of the solid phase carrier is preferably 0.3-0.5 mmol/g.

Further, the coupling agents for coupling in 1) and 3) are compositions of DIPCDI and HOBt, and the ratio of each component in the coupling agent to the amino acid raw material is, in terms of mass ratio, DIPCDI: HOBt: amino acid starting material 1.3: 1.2: 1.

further, the cleavage reagents for cleavage were TFA, TIS and H₂Composition of O, TFA, TIS, H₂The volume ratio of O is 90: 5: 5.

in addition, the introduction of Hmb to Val at position 23 using other oligopeptide fragments containing Hmb-Val residues will also be apparent to those skilled in the art.

The use of other framework protecting groups in place of the Hmb protecting groups, such as Hmsb, Hmnb, etc., is also readily contemplated by those skilled in the art.

The invention has the beneficial effects that: according to the invention, the Hmb protecting group is introduced into Val at position 23, so that beta folding in the process of peptide chain coupling can be effectively reduced, and further the reaction efficiency is improved, although skeleton protecting groups are introduced into other sites or the peptide chain skeleton can be changed by applying pseudo proline dipeptide, the price of amino acid raw materials with the skeleton protecting groups and the price of the pseudo proline dipeptide are expensive, the inventor finds that only the Hmb protecting group is introduced into Val at position 23 through a large amount of experimental screening and verification, so that the production cost is saved, a very obvious effect on improving beta folding is achieved, and the total yield and purity of the product are improved; the HPLC purity of the crude peptide T alpha l prepared by the method is more than 90 percent, generally can reach 98 percent, only has few impurities, greatly simplifies the difficulty of subsequent purification compared with the prior art, saves the purification cost and is beneficial to industrial application.

Drawings

FIG. 1 is a mass spectrum of a crude thymosin Talpha l peptide prepared by the method of the invention;

FIG. 2 is an HPLC chromatogram of the crude thymosin Talpha l peptide prepared by the present invention.

Detailed Description

For a better understanding of the present invention, the following examples are given to illustrate the present invention, but the present invention is not limited to the following examples.

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

abbreviations and English	Means of
		TFA	Trifluoroacetic acid
TIS	Tri-isopropyl silane
		Hmb	2 '-hydroxy-4' -methoxy-benzyl
Hmsb	2 ' -hydroxy-4 ' -methoxy-5 ' -methionyl-benzyl
		Hmnb	2 ' -hydroxy-4 ' -methoxy-5 ' -nitro-benzyl
HOBt	1-hydroxybenzotriazoles
		Fmoc	9-fluorenylmethoxycarbonyl group
DIPCDI	Diisopropylcarbodiimide
		DMF	N, N-dimethylformamide
DCM	Methylene dichloride
		tBu	Tert-butyl radical
OtBu	Tert-butoxy radical
		Dmb	2 ', 4' -dimethoxy-benzyl
Hnb	2 '-hydroxy-4' -nitro-benzyl

EXAMPLE 15 Synthesis of peptide resin

10g of Rink amide-MBHA resin with a degree of substitution of 0.5mmol/g was weighed, charged into a solid phase synthesis reactor, washed 2 times with DMF (50 mL each time, the same applies hereinafter), the solvent was removed after swelling the resin with DMF for 20 minutes, the resin was treated twice with 20% piperidine/DMF solution (5 minutes +7 minutes) to remove Fmoc, and the resin was washed 6 times with DMF. Weighing 6.17g/15mmol of Fmoc-Asp-OtBu and 2.43g/18mmol of HOBt, dissolving the solution in 35mL of DMF, adding 3mL/19.5mmol of DIPCDI, stirring for reaction for 3min, adding the solution into the reactor filled with the resin, pumping out the reaction solution after blowing nitrogen for 2 hours, washing the resin with DMF for 3 times, adding 20% piperidine/DMF solution to treat the resin twice (5 minutes +7 minutes) to remove the Fmoc group, and washing the resin with DMF for 6 times after the removal.

6.65g/15mmol of Fmoc-Glu (OtBu) -OH and 2.43g/18mmol of HOBt are weighed and dissolved in 35mL of DMF, 3mL/19.5mmol of DIPCDI is added and stirred for reaction for 3min, then the solution is added into the reactor filled with the resin, after 2 hours of reaction under nitrogen blowing, the reaction liquid is pumped out, the resin is washed by DMF for 3 times, 20% piperidine/DMF solution is added to treat the resin for two times (5 minutes +7 minutes) to remove the Fmoc group, and after the removal, the resin is washed by DMF for 6 times.

Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH and Fmoc-Glu (OtBu) -OH were coupled in sequence according to the same method to give DMF swollen 5-peptide resin for direct use in subsequent reactions.

EXAMPLE 2 preparation of 7 peptide resin-starting with Fmoc- (Fmoc-Hmb) -Val-OH

Using the DMF swollen 5 peptide resin prepared in example 1, 10.47g/15mmol of Fmoc- (Fmoc-Hmb) -Val-OH and 2.43g/18mmol of HOBt were weighed and dissolved in 35mL of DMF, 3mL/19.5mmol of DIPCDI was added and stirred for 3min, and after reaction, the solution was added to the above resin-filled reactor, the reaction solution was removed after 2 hours of nitrogen bubbling, the resin was washed with DMF 3 times, the resin was treated twice (5 min +7 min) with 20% piperidine/FmDMF solution (5 min +7 min) to remove the Fmoc group, and after removal, the resin was washed with DMF 6 times. Weighing 20.36g/60mmol of Fmoc-Val-OH, stirring uniformly with DCM, adding 5.4mL/35mmol of DIPCDI, stirring uniformly, adding the solid-liquid mixed solution into resin, reacting for 24h, removing the reaction solution, washing 3 times with DMF, 3 times with DCM and 3 times with methanol. After the solvent is pumped out, the 7-peptide resin is dried in vacuum, 15.62g is obtained, and the detection substitution degree is 0.21 mmol/g.

EXAMPLE 3 preparation of 7 peptide resin-starting with Fmoc-Val- (Fmoc-Hmb) -Val-OH

Using the DMF swollen 5 peptide resin prepared in example 1, 11.94g/15mmol of Fmoc- (Fmoc-Hmb) -Val-OH and 2.43g/18mmol of HOBt were weighed and dissolved in 35mL of DMF, 3mL/19.5mmol of DIPCDI was added and stirred for 3min, and after reaction, the solution was added to the above resin-filled reactor, after reacting for 2 hours under bubbling nitrogen, the reaction solution was removed, the resin was washed 3 times with DMF, 3 times with DCM and 3 times with methanol. After the solvent is pumped out, the 7-peptide resin is dried in vacuum, 15.88g is obtained, and the detection substitution degree is 0.29 mmol/g.

Example 4 preparation of 7-peptide resin-in situ introduction of Hmb by reductive amination

Using the DMF-swollen 5-peptide resin prepared in example 1, 2.28g/15mmol of p-methoxysalicylaldehyde was weighed, dissolved in 35mL of DMF, added to the resin, followed by addition of 0.35mL of glacial acetic acid, reaction under nitrogen bubbling for 1 hour, reaction solution was removed, and the resin was washed with DMF 3 times.

Weighing 0.57g/15mmol of sodium borohydride, dissolving the sodium borohydride in 35mL of DMF, adding the solution into a resin reactor, pumping out the reaction solution after 20 minutes, weighing 0.57g/15mmol of sodium borohydride, dissolving the sodium borohydride in 35mL of DMF, adding the solution into the resin reactor, and pumping out the reaction solution after 20 minutes. The resin was washed alternately with methanol and DMF 6 times and DCM three times.

Weighing 20.36g/60mmol of Fmoc-Val-OH, stirring uniformly with DCM, adding 5.4mL/35mmol of DIPCDI, stirring uniformly, adding the solid-liquid mixed solution into resin, reacting for 24h, removing the reaction solution, washing 3 times with DMF, 3 times with DCM and 3 times with methanol. After the solvent is pumped out, the 7 peptide resin is dried in vacuum, 15.13g is obtained, and the detection substitution degree is 0.22 mmol/g.

EXAMPLE 5 preparation of Talpha.l peptide resin

9.53g/2mmol of the 7-peptide resin prepared in example 2 was weighed, washed twice with DMF (40 mL each time, the same applies hereinafter) and then swollen with DMF for 20 minutes. The solvent was pumped off, the resin was treated twice (5 min +7 min) with 20% piperidine/DMF solution to remove Fmoc, and the resin was washed 6 times with DMF.

Weighing 2.66g/6mmol of Fmoc-Glu (OtBu) -OH and 0.97g/7.2mmol of HOBt, dissolving the solution in 30mL of DMF, adding 1.2mL/7.8mmol of DIPCDI, stirring for reaction for 3min, adding the solution into the reactor filled with the resin, pumping out the reaction solution after bubbling nitrogen for 2 hours, washing the resin with DMF for 3 times, treating the resin with 20% piperidine/DMF solution twice (5 minutes +7 minutes) to remove Fmoc groups, and washing the resin with DMF for 6 times after the removal.

Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Asp (tBu) -OH, Fmoc.

After Fmoc group removal, the resin was shrunk with methanol and dried under vacuum to give 16.22g of T.alpha.l peptide resin.

EXAMPLE 6 cleavage preparation of crude T.alpha.l peptide

16.22g of the T.alpha.l peptide resin obtained in example 5 was taken, and 150mL of a lysis solution (TFA: TIS: H) was added₂O (volume ratio) 90: 5: 5) the reaction was magnetically stirred for 2 hours. The resin was filtered off, the filtrate was poured into 1.2L of diethyl ether pre-frozen to about-5 ℃ to precipitate a white solid, which was centrifuged and the resulting solid was washed with diethyl ether 2 times. The obtained solid was purged with nitrogen to remove most of the solvent, and then vacuum-dried to obtain 5.2g of a white solid with a total yield of 84.8%. Mass spectrometric detection 3066.196, HPLC detection 98.43% purity.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.

Claims

1. A method for synthesizing thymosin T alpha-1 is characterized by comprising the following steps:

2) preparation of 7-peptide resin with Hmb protecting group:

NH₂-Val- (Hmb) Val-glu (otbu) -Ala-glu (otbu) - β -asp (otbu) -amino resin;

2. The method for synthesizing thymosin pha-1 of claim 1, wherein the preparation of said 5-peptide resin of 1) specifically comprises the following steps:

a. adding the solid phase carrier into a solid phase synthesis reactor, fully swelling with a solvent, removing the Fmoc protecting group with 20% piperidine/N, N-dimethylformamide solution, and washing;

b. adding Fmoc-Asp-OtBu and a coupling agent into N, N-dimethylformamide for reaction for a period of time, adding into a solid phase synthesis reactor, blowing nitrogen for reaction for a period of time, and washing; then removing Fmoc protecting groups by using 20% piperidine/N, N-dimethylformamide solution and washing; the above procedure was repeated to couple Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH and Fmoc-Glu (OtBu) -OH in this order.

3. The method for synthesizing thymosin pha-1 of claim 1, wherein said Hmb protecting group-bearing 7 peptide resin of 2) is prepared by the following method:

fmoc- (Fmoc-Hmb) -Val-OH is used as a raw material to be introduced, the 5-position Val is coupled by a symmetrical anhydride method after the Fmoc protecting group is removed after the 5-position peptide resin obtained in the step 1) is coupled with the Fmoc- (Fmoc-Hmb) -Val-OH according to the coupling method the same as that in the step 1), and 7-position peptide resin is obtained.

4. The method for synthesizing thymosin T α -1 of claim 1, wherein said Hmb protecting group-bearing 7-peptide resin of 2) is prepared by:

introducing a dipeptide fragment Fmoc-Val- (Fmoc-Hmb) -Val-OH serving as a raw material, coupling the Fmoc-Val- (Fmoc-Hmb) -Val-OH to the 5 peptide resin obtained in the step 1) according to the same coupling method as that in the step 1), and removing an Fmoc protecting group to obtain the 7 peptide resin.

5. The method for synthesizing thymosin T α -1 of claim 1, wherein said Hmb protecting group-bearing 7-peptide resin of 2) is prepared by:

forming a framework protecting group in situ through reductive amination reaction, treating the 5-peptide resin obtained in the step 1) with p-methoxysalicylaldehyde, reducing with sodium borohydride or sodium cyanoborohydride, washing, and coupling with 22-Val to obtain the 7-peptide resin.

6. The method for synthesizing thymosin T α -1 of claim 1, wherein said 28-peptide resin of 3) is prepared by the following steps:

a. adding the 7-peptide resin prepared in the step 2) into a solid phase synthesis reactor, fully swelling with a solvent, removing an Fmoc protecting group with 20% piperidine/N, N-dimethylformamide solution, and washing;

b. adding Fmoc-Glu (OtBu) -OH and a coupling agent into N, N-dimethylformamide for reacting for a period of time, adding into a solid phase synthesis reactor, reacting with nitrogen for a period of time, and washing; then removing Fmoc protecting groups by using 20% piperidine/N, N-dimethylformamide solution and washing; repeating the above process to couple Fmoc-Lys (Boc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ile-OH sequentially, Fmoc-Glu (OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -OH.

7. The method for synthesizing thymosin pha-1 of claim 1, wherein the solid phase carrier in 1) is selected from Rink Amide resin, Rink Amide-AM resin or Rink Amide-MBHA resin, and the substitution degree of the solid phase carrier is 0.1-0.7 mmol/g.

8. The method for synthesizing thymosin pha-1 of claim 7, wherein the degree of substitution of said solid phase carrier is 0.3-0.5 mmol/g.

9. The method for synthesizing thymosin pha-1 of claims 1-6, wherein said coupling agent for coupling in 1) and 3) is a composition of DIPCDI and HOBt, and the ratio of each component in said coupling agent to amino acid raw material is DIPCDI: HOBt: amino acid starting material 1.3: 1.2: 1.

10. the method for synthesizing thymosin T α -1 of any one of claims 1-6, wherein said cleavage reagent for cleavage is TFA, TIS and H₂O, said TFA, TIS, H₂The volume ratio of O is 90: 5: 5.