JP2680446B2 - Solid phase peptide synthesis - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to peptide synthesis useful in the development of a heptid drug, a synthetic vaccine, the determination of the antigenic site of a protein, the preparation of an antibody, etc., particularly in the solid phase method. The present invention relates to improvements in peptide synthesis methods.

[Prior Art] Many natural polypeptides have a specific physiological activity, and some of them function as important drugs.
However, when it is obtained from nature, it is extremely difficult to obtain a large amount because of its low content and inefficient extraction.
Chemical synthesis of polypeptides has been actively carried out. There are “liquid phase method” and “solid phase method” in the chemical synthesis of polypeptide, and in recent years, the solid phase method has become the mainstream because of the simplicity that the purification operation after each reaction step only requires washing of the resin. Has become.

Solid phase peptide synthesis is generally performed by the following method. First, an amino acid or peptide whose N-terminal is protected by a protecting group is taken out and this C-terminal is fixed to a resin. Then, the protective group is removed by washing with an acid or an alkali. Furthermore, it is condensed with an amino acid or peptide whose N-terminus is protected. Then, a ninhydrin test is performed to check whether the condensation is completed, the condensation is repeated until the test becomes negative, washing is performed, and then the condensation is sequentially repeated. By repeating this cycle, n amino acids are synthesized. After introducing all the peptide chains, the bond with the resin is cleaved and separated from the resin to obtain the desired polypeptide.

The major problem with this method is that if the number of reactions increases and the yield after one reaction is insufficient, if unreacted polypeptide remains, another peptide chain will grow due to the next reaction. I will end up. By repeating this process, a poorly pure peptide chain is finally obtained. In this case, the peptide chain as an impurity has a structure similar to that of the expected peptide chain, which makes purification very difficult.

Therefore, in order to obtain a highly pure polypeptide, 1
It is necessary to complete the reaction once, and various studies have been made on the reaction conditions, condensing agent, etc., but until now, it has been difficult to complete the condensation particularly in the polypeptide synthesis system, and a pure polypeptide is obtained. It wasn't easy.

[Means for Solving Problems] As a result of intensive studies made by the present inventors in view of the problems in the conventional methods, they found that this object can be achieved by incorporating a specific reaction solvent into a reaction system. The present invention has been reached. That is, in the present invention, a C-terminal amino acid is immobilized on an insoluble resin, and in the solid-phase peptide synthesis method in which the amino acid or peptide is sequentially condensed on the resin to extend the peptide chain, a condensation reaction is performed and then a reaction solvent is used. To 1 to 5% by volume of hexafluoroisopropyl alcohol to complete the reaction.

The method of the present invention can be applied to both the t-Boc method and the Fmoc method, and various commercially available resins can be used as the insoluble resin used in the reaction, and may be selected according to the target polypeptide. . In the reaction, the peptide resin should be deprotected with a solution such as TFA / CH ₂ Cl ₂ as usual, washed, neutralized with a base such as Et ₃ N, if necessary, and then added. Reacts with carboxy-activators of amino acids or peptides. As the reaction solvent used in this reaction, the base methylene is most preferably used. Alternatively, the peptide insoluble in methylene chloride is reacted, or hexafluoroisopropyl alcohol (hereinafter referred to as HFIP) in the range of 1 to 5% by volume from the beginning is added to dissolve the peptide to carry out the reaction. Whether or not the condensation reaction is completed is checked by a ninhydrin test, and if not negative, the condensation reaction is further carried out using a mixed solvent of HFIP and a chlorinated hydrocarbon solvent as a reaction solvent. The amount of HFIP is chosen to be 1-5% by volume of HFIP. Below this range, it does not contribute to completion of the condensation reaction, and above this range, side reactions proceed, which is not preferable.

In the method of the present invention, it is not preferable to use a mixed solvent of HFIP at the start of the condensation reaction except when the peptide chloride is insoluble in methylene. This is because if a HFIP mixed solvent is used at the start of the reaction, the hydroxyl group of HFIP reacts with the carboxylic acid to generate an ester byproduct. On the other hand, when the mixed solvent of the present invention is added in the final stage of the condensation reaction, the above esterification also proceeds, but there is no particular problem in completing the condensation reaction, but in this case, a carboxylic acid or the like is newly added. That is also effective.

 Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 (Crosslinked polystyrene support) 0.8g (Peptide content 0.094m
Mol) was swollen in a mixed solvent of TFA / CH ₂ Cl ₂ (2: 3) 15 ml, stirred at room temperature for 2 hours, and deprotected. The resin was filtered through a glass filter, washed with methanol and CH ₂ CL ₂ alternately, and finally with CH ₂ Cl ₂ thoroughly, then the resin was transferred to a reaction vessel and swollen with 20 ml of CH ₂ Cl _2. Et ₃ N 0.19 g (20 eq) was added and the mixture was stirred for 10 minutes for neutralization. The resin was filtered through a glass filter, washed alternately with methanol and CH ₂ Cl ₂ , and finally thoroughly washed with CH ₂ Cl ₂ before being transferred to the resin and reaction vessel and swollen in 14 ml of CH ₂ Cl _2. , To which Boc-X-OH (0.094
mmol × 3), 1-hydroxybenzotriazole (0.09
4 mmol × 3) and dicyclohexylcarbodiimide (0.094 mmol × 3) were added and reacted at room temperature for 15 hours.
When the ninhydrin test is positive, the same reaction is repeated, and then 1 to 5% with respect to the reaction solvent CH ₂ Cl ₂ .
(Volume ratio) The addition of hexafluoroisopropanol condensation reaction is terminated, the ninhydrin test becomes _{negative, Boc-X 2 -Val-Ile} -NH-CH 2 are obtained, further Bo
instead of Boc-X-OH relative to _{c-X 2 -Val-Ile-} NH-CH 2 by reacting Boc-Val-Ile-OH Was obtained, and the reaction operation with Boc-X-OH was performed twice, was gotten. The amino acid analysis value of the obtained resin was in good agreement with the theoretical value. The amino acid residue X is Ala or Arg.
(Tos), Asn, AsP (OBz1), Cys (Bz1), G1n, G1u
(OBz1), G1y, His (Tos), I1e, Leu, Lys (Z), Me
t (0), Phe, Ser (Bz1), Thr (Bz1), Trp (CHO),
Tyr (Bz1) and Va1 were used.

Comparative Example 1 CH ₂ C ₁₂ was used as the reaction solvent in the same manner as in Example 1, but the ninhydrin test did not become negative forever.

[Effect of the Invention] According to the method of the present invention, various polypeptides can be easily obtained with high yield and high purity.

Claims (1)

(57) [Claims] 1. A solid phase peptide synthesis method in which a C-terminal amino acid is immobilized on an insoluble resin, and the amino acid or peptide is sequentially condensed on the resin to extend the peptide chain, and a condensation reaction is carried out, followed by a reaction solvent. For 1 to 5
A solid-phase peptide synthesis method, which comprises adding hexafluoroisopropyl alcohol in a volume range to complete the reaction.