KR20210104460A - A Method for preparing peptides using by solid phase synthesis - Google Patents

A Method for preparing peptides using by solid phase synthesis Download PDF

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KR20210104460A
KR20210104460A KR1020200019233A KR20200019233A KR20210104460A KR 20210104460 A KR20210104460 A KR 20210104460A KR 1020200019233 A KR1020200019233 A KR 1020200019233A KR 20200019233 A KR20200019233 A KR 20200019233A KR 20210104460 A KR20210104460 A KR 20210104460A
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fmoc
gly
resin
mmol
peptide
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양재욱
임형준
임택주
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주식회사 아이바이오코리아
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Priority to PCT/KR2021/001959 priority patent/WO2021167317A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C07K1/122—Hydrolysis with acids different from HF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

The present invention provides a method for preparing a peptide represented by the sequence of structural formula (a), the method includes: a first step of using a reaction solvent to a resin into which a trityl group is introduced so as to sequentially couple amino acid derivatives, in which both amine terminals and side chains are protected; and a second step of separating a peptide from the resin of the first step and removing side chain protecting groups. Structural formula (a) Hyp-Gly-Gln-Asp-Gly-Leu-Ala-Gly-Pro-Lys. Therefore, peptide synthesis can be performed more conveniently than solution-phase synthesis.

Description

고체상 합성법을 이용한 펩타이드의 제조 방법{A Method for preparing peptides using by solid phase synthesis}A method for preparing a peptide using a solid phase synthesis method {A Method for preparing peptides using by solid phase synthesis}

본 발명의 다양한 실시예는 안구 표면질환, 황반변성 및 건선안을 예방하거나 치료할 수 있는 펩타이드의 제조 방법에 관한 것이다. Various embodiments of the present invention relate to a method for producing a peptide capable of preventing or treating ocular surface disease, macular degeneration, and psoriatic eye.

펩타이드를 화학적으로 합성하는 방법은 크게 용액상 합성법과 고체상 합성법으로 나눌 수 있다. 용액상 합성법은 고전적인 화학 합성법으로 모든 시약을 용액에 녹인 상태에서 반응시키는 방법으로 반응 속도는 빠르지만 분리 정제가 어렵다는 단점이 있다. 고체상 합성법은 메리필드(R. B. Merrifield)가 고체상 펩타이드 합성(solid phase peptide synthesis)에 관한 이론을 제기한 이래, 발전해온 방법으로 분리정제가 간편하며 자동화가 가능하다는 장점이 있다(Bodanszky et al, In Peptide Synthesis, JohnWiley & Sons, 1976).Methods for chemically synthesizing peptides can be largely divided into solution phase synthesis methods and solid phase synthesis methods. The solution-phase synthesis method is a classical chemical synthesis method in which all reagents are dissolved in a solution. Although the reaction rate is fast, it has a disadvantage in that separation and purification are difficult. The solid phase synthesis method has been developed since RB Merrifield proposed a theory on solid phase peptide synthesis, and has the advantage of simple separation and purification and automation (Bodanszky et al, In Peptide). Synthesis, John Wiley & Sons , 1976).

메리필드(Merrifield)의 연구 이후 수많은 펩타이드 합성용 수지들이 개발되어 왔고 또 이를 이용하여 많은 펩타이드들이 합성되어 왔다. 메리필드에 의하여 개발된 클로로메틸 폴리스티렌(chloromethyl polystyrene)수지, 그리고 이 수지의 단점을 보완하여 개발된 4-알콕시벤질 알콜(4-alkoxybenzyl alcohol)의 구조를 갖는 왕(Wang) 수지가 비교적 초기에 개발되었다. 이후 이들의 단점을 보완하는 수지들이 지속적으로 개발되어 왔는데, 그 중 트리틸 구조가 도입된 2-클로로트리틸 수지와 펩타이드의 카르복실 말단을 아미드 형태로 얻을 수 있는 링크아미드 수지가 대표적 수지이다.After the research of Merrifield, numerous resins for peptide synthesis have been developed and many peptides have been synthesized using them. Chloromethyl polystyrene resin developed by Maryfield, and Wang resin having a structure of 4-alkoxybenzyl alcohol developed by supplementing the disadvantages of this resin were developed relatively early. became Since then, resins that compensate for these disadvantages have been continuously developed. Among them, 2-chlorotrityl resin having a trityl structure and linkamide resin capable of obtaining the carboxyl terminus of a peptide in the form of an amide are representative resins.

이들 수지를 이용하여 초기에는 간단한 펩타이드들이 합성되었으나, 최근에는 생리활성을 갖는 복잡한 펩타이드들 합성이 요구되고 있다. 그러나, 이러한 복잡한 구조의 펩타이드 합성 시 만족할 만한 순도의 물질을 얻기가 어려워 이를 해결하기 위한 제조 방법의 개발이 요구되고 있다.Simple peptides were initially synthesized using these resins, but recently, the synthesis of complex peptides having physiological activity is required. However, it is difficult to obtain a material of satisfactory purity when synthesizing a peptide having such a complex structure, and development of a manufacturing method is required to solve this problem.

이에 본 발명자들은 아민 말단 및 측쇄가 모두 보호된 아미노산 유도체들을 고분자 지지체와 순차적으로 결합시켜 펩타이드를 제조하는 방법으로, 안구 표면질환, 황반변성 및 건선안을 예방하거나 치료할 수 있는 펩타이드를 고순도 및 고수율로 제조할 수 있음을 발견하여 본 발명을 완성하였다.Accordingly, the present inventors are a method for preparing a peptide by sequentially combining amino acid derivatives with both protected amine terminus and side chains with a polymer support, and a peptide capable of preventing or treating ocular surface disease, macular degeneration and psoriasis with high purity and high yield. The present invention was completed by finding that it can be prepared.

본 발명은, 트리틸기가 도입된 수지에 반응용매를 이용하여 아민말단 및 측쇄가 모두 보호된 아미노산 유도체들을 순차적으로 결합시키는 제1 단계; 및 상기 제1 단계의 수지에서 펩타이드를 분리하고 측쇄 보호기들을 제거하는 제2 단계의 공정을 수행함을 특징으로 하는 하기 구조식 (a)의 서열로 표시되는 펩타이드 제조 방법을 제공한다.The present invention provides a first step of sequentially binding amino acid derivatives in which both the amine terminus and the side chain are protected using a reaction solvent to a resin having a trityl group introduced thereto; And it provides a method for producing a peptide represented by the sequence of Structural Formula (a) below, characterized in that the second step of separating the peptide from the resin of the first step and removing the side chain protecting groups is performed.

구조식 (a) Hyp-Gly-Gln-Asp-Gly-Leu-Ala-Gly-Pro-LysStructural formula (a) Hyp-Gly-Gln-Asp-Gly-Leu-Ala-Gly-Pro-Lys

본 발명의 고체상 펩타이드를 제조하는 방법에 관한 것으로, 트리틸기가 도입된 수지에 아미노산 유도체들을 순차적으로 반응시켜 펩타이드 수지를 얻고 이 수지에서 펩타이드를 최종적으로 유리시켜 펩타이드를 제조하는 방법으로, 전 제조 공정을 고체상 지지체 상에서 실시하므로 펩타이드 합성을 용액상 합성법보다 간편하게 할 수 있으며, 종래 기술의 문제점인 고비용 및 소량 생산 등의 단점을 개선하여 목적물질을 고수율 및 저비용으로 대량 생산이 가능하도록 하였다. The present invention relates to a method for producing a solid-phase peptide, in which amino acid derivatives are sequentially reacted with a resin having a trityl group introduced thereto to obtain a peptide resin, and the peptide is finally released from the resin to prepare a peptide, the entire manufacturing process is carried out on a solid-phase support, so that peptide synthesis can be made simpler than solution-phase synthesis, and disadvantages such as high cost and small-volume production, which are problems of the prior art, are improved to enable mass production of the target material at high yield and low cost.

이하, 본 문서의 다양한 실시예들이 첨부된 도면을 참조하여 기재된다. 실시예 및 이에 사용된 용어들은 본 문서에 기재된 기술을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 해당 실시예의 다양한 변경, 균등물, 및/또는 대체물을 포함하는 것으로 이해되어야 한다. Hereinafter, various embodiments of the present document will be described with reference to the accompanying drawings. The examples and terms used therein are not intended to limit the technology described in this document to specific embodiments, and should be understood to include various modifications, equivalents, and/or substitutions of the embodiments.

본 발명은, 트리틸기가 도입된 수지에 반응용매를 이용하여 아민말단 및 측쇄가 모두 보호된 아미노산 유도체들을 순차적으로 결합시키는 제1 단계; 및 상기 제1 단계의 수지에서 펩타이드를 분리하고 측쇄 보호기들을 제거하는 제2 단계의 공정을 수행함을 특징으로 하는 하기 구조식 (a)의 서열로 표시되는 펩타이드 제조 방법을 제공한다.The present invention provides a first step of sequentially binding amino acid derivatives in which both the amine terminus and the side chain are protected using a reaction solvent to a resin having a trityl group introduced thereto; And it provides a method for producing a peptide represented by the sequence of Structural Formula (a) below, characterized in that the second step of separating the peptide from the resin of the first step and removing the side chain protecting groups is performed.

구조식 (a) Hyp-Gly-Gln-Asp-Gly-Leu-Ala-Gly-Pro-LysStructural formula (a) Hyp-Gly-Gln-Asp-Gly-Leu-Ala-Gly-Pro-Lys

구조식 (a)의 서열에서, Hyp는 하이드록시 프롤린(Hydroxy proline), Gly는 글라이신(Glycine), Gln은 글루타민(Glutamine), Asp는 아스파트산(Aspartic acid), Leu는 류신(Leucine), Ala는 알라닌(Alanine), Pro는 프롤린(Proline), Lys는 라이신(Lysine)을 의미한다. In the sequence of structural formula (a), Hyp is hydroxy proline, Gly is glycine, Gln is glutamine, Asp is aspartic acid, Leu is leucine, Ala stands for Alanine, Pro stands for Proline, and Lys stands for Lysine.

한편, 아미노산 유도체는 아민말단 및 측쇄가 Boc(tert-butoxycarbonyl), Fmoc(9-fluorenylmethoxycarbonyl), OtBu(tert-Butoxy), tBu(tert-Butyl) 및 Trt(Trityl)로 이루어진 군에서 선택된 어느 하나의 보호기로 보호될 수 있다. On the other hand, the amino acid derivative has an amine terminus and side chain selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), OtBu (tert-Butoxy), tBu (tert-Butyl) and Trt (Trityl). It can be protected with a protecting group.

본 발명의 반응용매로는 디클로메탄, 클로로포름, 디클로로에탄, 디메틸포름아마드, 디메틸아세트아미드, N-메틸피로리디논, 테트라히드로푸란, 트리플루오르아세트산, 디옥산 또는 이들의 혼합용매로부터 선택될 수 있다. The reaction solvent of the present invention may be selected from dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, N-methylpyrrolidinone, tetrahydrofuran, trifluoroacetic acid, dioxane, or a mixture thereof. have.

본원에 개시된 반응들의 반응 온도는 그 제한은 없으나, 바람직하게는 0 ℃ 내지 70 ℃, 보다 바람직하게는 20 ℃ 내지 50 ℃의 범위이다. 반응시간은 10 분 내지 48 시간의 범위이며, 바람직하게는 각 반응물질의 반응성과 반응 후 생성물의 생산성을 고려할 때, 1 시간 내지 24 시간의 범위이다. 단, 원하는 만큼 반응이 진행되지 않았을 경우에는 동일 반응을 2 회 내지 5 회 더 수행하여 반응 수율을 높일 수 있다.The reaction temperature of the reactions disclosed herein is not limited thereto, but preferably ranges from 0°C to 70°C, more preferably from 20°C to 50°C. The reaction time is in the range of 10 minutes to 48 hours, preferably in the range of 1 hour to 24 hours in consideration of the reactivity of each reactant and the productivity of the product after the reaction. However, when the reaction has not progressed as much as desired, the same reaction may be performed 2 to 5 more times to increase the reaction yield.

본 발명의 목적은 안구 표면질환, 황반변성 및 건선안을 예방하거나 치료할 수 있는 구조식 (a)의 서열을 갖는 펩타이드의 제조 방법으로, 보다 구체적으로 다음과 같은 단계들에 의해 제조될 수 있다. An object of the present invention is a method for preparing a peptide having the sequence of Structural Formula (a) that can prevent or treat ocular surface disease, macular degeneration and psoriatic eye, and more specifically, it can be prepared by the following steps.

본 발명의 구조식 (a)의 서열을 갖는 펩타이드의 제조 방법은, 2-염화 클로로트리틸(Chlorotrityl chloride) 형 수지를 준비하는 단계; 상기 수지에 반응용매로 Fmoc기를 제거하여 탈보호 반응을 수행하는 단계; 상기 반응물에 Fmoc-Pro, Fmoc-Gly, Fmoc-Ala, Fmoc-Leu, Fmoc-Gly, Fmoc-Asp(OtBu), Fmoc-Gln(Trt), Fmoc-Gly, 및 Fmoc-Hyp(tBu)을 순차적으로 첨가하여 커플링 반응시키는 단계; 상기 반응물의 Fmoc기를 탈보호하는 단계; 상기 반응물에서 수지를 제거하여 상기 구조식(a)의 서열로 표기되는 펩타이드를 수득하는 단계; 및 상기 펩타이드를 정제하는 단계를 포함할 수 있다.The method for producing a peptide having the sequence of Structural Formula (a) of the present invention comprises the steps of preparing a 2-chloride chlorotrityl (Chlorotrityl chloride) type resin; performing a deprotection reaction by removing the Fmoc group from the resin as a reaction solvent; Fmoc-Pro, Fmoc-Gly, Fmoc-Ala, Fmoc-Leu, Fmoc-Gly, Fmoc-Asp(OtBu), Fmoc-Gln(Trt), Fmoc-Gly, and Fmoc-Hyp(tBu) were sequentially added to the reaction. adding to the coupling reaction; deprotecting the Fmoc group of the reactant; removing the resin from the reaction to obtain a peptide represented by the sequence of the structural formula (a); and purifying the peptide.

이때, 2-염화 클로로트리틸(chlorotrityl chloride) 형 수지에 Fmoc-Lys(Boc)를 붙인 수지는 2-염화 클로로트리틸 형 링크-라이신형 수지(link-Lys-Resin)이다. 2-염화 클로로트리틸 형 링크-라이신형 수지에서 링크(Link)는 하기 화학식 1과 같은 구조를 갖는 링크 2-클로로트리틸 클로라이드(2- chlorotrityl chloride)임을 특징으로 한다.At this time, the 2-chloride chlorotrityl (chlorotrityl chloride) type resin to which Fmoc-Lys (Boc) is attached is a 2-chlorotrityl type link-lysine type resin (link-Lys-Resin). The link in the 2-chlorinated chlorotrityl-type link-lysine-type resin is characterized in that the link is 2-chlorotrityl chloride having a structure as shown in Chemical Formula 1 below.

[화학식 1][Formula 1]

Figure pat00001
Figure pat00001

2-염화 클로로트리틸 형 링크-라이신형 수지에 반응용매를 이용하여 Fmoc기를 제거하여 탈보호 반응을 수행하는 단계에서 반응용매는, 피페리딘(piperidine) 또는 DMF(dimethyl formamide)일 수 있다. 바람직하게는, 20 % 피페리딘을 사용하여 Fmoc 기를 제거하여 탈보호 반응이 진행될 수 있다. In the step of performing the deprotection reaction by removing the Fmoc group from the 2-chlorotrityl linkage-lysine-type resin using a reaction solvent, the reaction solvent may be piperidine or dimethyl formamide (DMF). Preferably, the deprotection reaction can proceed by removing the Fmoc group using 20% piperidine.

Fmoc기가 제거된 반응물에 Fmoc-Pro, Fmoc-Gly, Fmoc-Ala, Fmoc-Leu, Fmoc-Gly, Fmoc-Asp(OtBu), Fmoc-Gln(Trt), Fmoc-Gly, 및 Fmoc-Hyp(tBu)을 순차적으로 첨가하여 커플링 반응시킬 수 있다. 즉, 아민 말단 및 측쇄가 모두 보호된 아미노산 유도체들을 순차적으로 결합시킬 수 있다. 이때, 각 아미노산 유도체를 결합시킨 후 다음 아미노산 유도체를 결합시키기 전에 Fmoc 기를 탈보호할 수 있다. Fmoc-Pro, Fmoc-Gly, Fmoc-Ala, Fmoc-Leu, Fmoc-Gly, Fmoc-Asp(OtBu), Fmoc-Gln(Trt), Fmoc-Gly, and Fmoc-Hyp(tBu) ) can be sequentially added for a coupling reaction. That is, amino acid derivatives in which both the amine terminus and the side chain are protected can be sequentially linked. In this case, after binding each amino acid derivative, the Fmoc group may be deprotected before binding to the next amino acid derivative.

한편, 상기 커플링 반응시키는 단계에서 사용되는 커플링 시약은 각 아미노산의 활성 에스테르, DCC(dicyclohexyl carbodiimide), DIC(diisopropyl carbodiimide), BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP(Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HBTU(O-Benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate), TBTU(O-(Benzotriaol-1-yl)- N,N,N',N'-tetramethyl-uronium tetrafluoroborate), HATU(2-(1H-7-Azabenzotriaol-1-yl)-1,1,3,3- tetramethyl uranium hexafluorophosphate Methanaminium), TATU(2-(1H-7-Azabenzotriaol-1-yl)- 1,1,3,3- tetramethyl uranium tetrafluoroborate Methanaminium) 및 CDI(carbonyl diimidazole)로 이루어진 군에서 선택된 적어도 어느 하나일 수 있다. 바람직하게는 DIC(diisopropyl carbodiimide)를 이용하여 커플링 반응시킬 수 있다. 커플링 시약의 사용량은 아미노산 당량에 대해 약 0.5 내지 10 당량, 바람직하게는 약 0.5 내지 3 당량일 수 있다. 아미노산 유도체의 양은 수지의 치환율에 대해 약 1 내지 10 당량, 바람직하게는 약 1.5 내지 3 당량일 수 있다. 상기한 아미노산 활성 에스테르로는 아미노산 유도체의 대칭 무수화물(symmetric anhydride), 혼합 무수화물(mixed anhydride), 펜타플루오르 페닐 에스테르 등 아미노산의 C 말단을 활성화시키는 물질로서 고분자 수지에 결합된 아미노산 당량에 대해 약 1 내지 10 당량 사용하는 것이 바람직하다.On the other hand, the coupling reagent used in the coupling reaction step is active ester of each amino acid, DCC (dicyclohexyl carbodiimide), DIC (diisopropyl carbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium) hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate), TBTU (O-(Benzotriaol-1-yl)-N, N,N',N'-tetramethyl-uronium tetrafluoroborate), HATU(2-(1H-7-Azabenzotriaol-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate Methanaminium), TATU(2-(1H) -7-Azabenzotriaol-1-yl)- 1,1,3,3-tetramethyl uranium tetrafluoroborate Methanaminium) and CDI (carbonyl diimidazole) may be at least one selected from the group consisting of. Preferably, the coupling reaction may be performed using diisopropyl carbodiimide (DIC). The amount of the coupling reagent used may be about 0.5 to 10 equivalents, preferably about 0.5 to 3 equivalents, based on the equivalent of amino acid. The amount of the amino acid derivative may be about 1 to 10 equivalents, preferably about 1.5 to 3 equivalents, based on the substitution rate of the resin. The amino acid active ester described above is a substance that activates the C-terminus of amino acids such as symmetric anhydride, mixed anhydride, and pentafluorphenyl ester of amino acid derivatives. It is preferable to use 1 to 10 equivalents.

한편, 펩타이드를 수득하는 단계에서는, 최종 반응물에 강산성 분해(cleavage) 용액을 첨가하여 최종 반응물에서 펩타이드를 떼어내는 것을 특징으로 한다. 강산성 분해 용액은 TFA(trifluoroacetic acid) 또는 DCM(dichloromethane) 일 수 있다. 강산성 분해 용액은, 펩타이드 수지에 대해 2 % 내지 50 % 포함될 수 있다. Meanwhile, in the step of obtaining the peptide, a strong acid cleavage solution is added to the final reactant to separate the peptide from the final reactant. The strongly acidic decomposition solution may be trifluoroacetic acid (TFA) or dichloromethane (DCM). The strongly acidic decomposition solution may be included in an amount of 2% to 50% with respect to the peptide resin.

펩타이드를 정제하는 단계에서는, 펩타이드를 역상 컬럼(LC)으로 정제하고, 이온교환 수지를 이용하여 정제할 수 있다. 이를 통해 본 발명의 펩타이드를 아세테이트 염의 형태로 수득할 수 있다. In the step of purifying the peptide, the peptide may be purified by a reverse phase column (LC) and purified using an ion exchange resin. Through this, the peptide of the present invention can be obtained in the form of an acetate salt.

본 발명의 펩타이드 제조 방법은, 트리틸기가 도입된 수지에 아미노산 유도체들을 순차적으로 반응시켜 펩타이드 수지를 얻고 이 수지에서 펩타이드를 최종적으로 유리시켜 펩타이드를 제조하는 방법으로, 전 제조 공정을 고체상 지지체 상에서 실시하므로 펩타이드 합성을 용액상 합성법보다 간편하게 할 수 있으며, 종래 기술의 문제점인 고비용 및 소량 생산 등의 단점을 개선하여 목적물질을 고수율 및 저비용으로 대량 생산이 가능하다. The peptide production method of the present invention is a method for producing a peptide by sequentially reacting amino acid derivatives with a resin having a trityl group introduced thereto to obtain a peptide resin and finally releasing the peptide from the resin. Therefore, peptide synthesis can be made simpler than solution-phase synthesis, and disadvantages such as high cost and small volume production, which are problems of the prior art, can be improved to mass-produce the target substance at high yield and low cost.

이하, 구체적인 실시예는 다음과 같다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 여기에 제한되거나 한정되고자 함은 아니다.Hereinafter, specific examples are as follows. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not intended to be limited or limited thereto.

실시예 1: Trityl resin-Lys(Boc)-Fmoc의 제조Example 1: Preparation of Trityl resin-Lys(Boc)-Fmoc

2- Chlorotrityl resin (150 g, 219 mmol)에 MC (10 v/mmol, 2.2 L) 주입하여 1 시간 교반한 후 탈수한다. 다른 반응부에 Fmoc-Lys(Boc)-OH (205.22 g, 438 mmol)과 MC (8 v/mmol, 1.8 L), DIEA (153 ml, 876 mmol) 주입하여 10 분 교반하여 용해한 후 resin이 들어있는 반응부에 주입하여 상온에서 4 시간 교반한다. 교반 후 탈수한다. 2- Chlorotrityl resin (150 g, 219 mmol) is injected with MC (10 v/mmol, 2.2 L), stirred for 1 hour, and then dehydrated. Fmoc-Lys(Boc)-OH (205.22 g, 438 mmol), MC (8 v/mmol, 1.8 L), and DIEA (153 ml, 876 mmol) were injected into the other reaction part, stirred for 10 minutes to dissolve, and then the resin was added It is injected into the reaction part and stirred at room temperature for 4 hours. After stirring, dehydrate.

실시예 2: Trityl resin-Lys(Boc)-Pro-Fmoc의 제조Example 2: Preparation of Trityl resin-Lys(Boc)-Pro-Fmoc

실시예 1에서 합성된 Trityl resin-Lys(Boc)-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Pro-OH (9.5 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Fmoc을 수득할 수 있다.After injecting 20% piperidine (8 v/mmol, 113 ml) into Trityl resin-Lys(Boc)-Fmoc (14.09 mmol) synthesized in Example 1, stir for 10 minutes to dehydrate. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Pro-OH (9.5 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, it is stirred at room temperature for 4 hours and dehydrated to obtain the desired Trityl resin-Lys(Boc)-Pro-Fmoc.

실시예 3: Trityl resin-Lys(Boc)-Pro-Gly-Fmoc의 제조Example 3: Preparation of Trityl resin-Lys(Boc)-Pro-Gly-Fmoc

실시예 2에서 합성된 Trityl resin-Lys(Boc)-Pro-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Gly-OH (8.38 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Fmoc을 수득할 수 있다.After injecting 20% piperidine (8 v/mmol, 113 ml) into Trityl resin-Lys(Boc)-Pro-Fmoc (14.09 mmol) synthesized in Example 2, stir for 10 minutes to dehydrate. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Gly-OH (8.38 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, it is stirred at room temperature for 4 hours and dehydrated to obtain the desired Trityl resin-Lys(Boc)-Pro-Gly-Fmoc.

실시예 4: Trityl resin-Lys(Boc)-Pro-Gly-Ala-Fmoc의 제조Example 4: Preparation of Trityl resin-Lys(Boc)-Pro-Gly-Ala-Fmoc

실시예 3에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Ala-OH (8.77 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Fmoc을 수득할 수 있다.After injecting 20% piperidine (8 v/mmol, 113 ml) into Trityl resin-Lys(Boc)-Pro-Gly-Fmoc (14.09 mmol) synthesized in Example 3, stir for 10 minutes to dehydrate. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Ala-OH (8.77 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, it is stirred at room temperature for 4 hours and dehydrated to obtain the desired Trityl resin-Lys(Boc)-Pro-Gly-Ala-Fmoc.

실시예 5: Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Fmoc의 제조Example 5: Preparation of Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Fmoc

실시예 4에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Leu-OH (9.96 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Fmoc을 수득할 수 있다.After injecting 20% piperidine (8 v/mmol, 113 ml) into Trityl resin-Lys(Boc)-Pro-Gly-Ala-Fmoc (14.09 mmol) synthesized in Example 4, stir for 10 minutes to dehydrate. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Leu-OH (9.96 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, it is stirred at room temperature for 4 hours and dehydrated to obtain the desired Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Fmoc.

실시예 6: Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Fmoc의 제조Example 6: Preparation of Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Fmoc

실시예 5에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Gly-OH (8.38 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Fmoc을 수득할 수 있다.After injecting 20% piperidine (8 v/mmol, 113 ml) into Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Fmoc (14.09 mmol) synthesized in Example 5, it is dehydrated by stirring for 10 minutes. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Gly-OH (8.38 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, it is stirred at room temperature for 4 hours and dehydrated to obtain the desired Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Fmoc.

실시예Example 7: 7: TritylTrityl resin- resin- LysLys (( BocBoc )-Pro-)-Pro- GlyGly -Ala-Leu--Ala-Leu- GlyGly -Asp(-Asp( OtBuOtBu )-)- Fmoc의Fmoc's 제조 Produce

실시예 6에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Asp(OtBu)-OH (11.59 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Fmoc을 수득할 수 있다.Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Fmoc (14.09 mmol) synthesized in Example 6 was injected with 20% piperidine (8 v/mmol, 113 ml) and stirred for 10 minutes to dehydrate. . Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Asp(OtBu)-OH (11.59 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. DIC (28.18 mmol, 4.4 ml) was injected into the main reaction part, stirred at room temperature for 4 hours, and dehydrated to obtain the desired Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Fmoc can do.

실시예Example 8: 8: TritylTrityl resin- resin- LysLys (( BocBoc )-Pro-)-Pro- GlyGly -Ala-Leu--Ala-Leu- GlyGly -Asp(-Asp( OtBuOtBu )-)- GlnGln (( TrtTrt )-Fmoc의 제조)-Fmoc preparation

실시예 7에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Gln(Trt)-OH (17.2 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Fmoc을 수득할 수 있다.After injection of 20% piperidine (8 v/mmol, 113 ml) into Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Fmoc (14.09 mmol) synthesized in Example 7, 10 Dehydrate by stirring for minutes. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Gln(Trt)-OH (17.2 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, stir at room temperature for 4 hours and dehydrate to the desired Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt) )-Fmoc can be obtained.

실시예Example 9: 9: TritylTrityl resin- resin- LysLys (( BocBoc )-Pro-)-Pro- GlyGly -Ala-Leu--Ala-Leu- GlyGly -Asp(-Asp( OtBuOtBu )-)- GlnGln (( TrtTrt )-Gly-Fmoc의 제조)-Gly-Fmoc preparation

실시예 8에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Gly-OH (8.38 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Gly-Fmoc을 수득할 수 있다.20% piperidine (8 v/mmol, 113) in Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Fmoc (14.09 mmol) synthesized in Example 8 ml) after injection and stirred for 10 minutes to dehydrate. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Gly-OH (8.38 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, stir at room temperature for 4 hours and dehydrate to the desired Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt) )-Gly-Fmoc can be obtained.

실시예Example 10: 10: TritylTrityl resin- resin- LysLys (( BocBoc )-Pro-)-Pro- GlyGly -Ala-Leu--Ala-Leu- GlyGly -Asp(-Asp( OtBuOtBu )-Gln(Trt)-Gly-Hyp-Fmoc의 제조Preparation of )-Gln(Trt)-Gly-Hyp-Fmoc

실시예 9에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Gly-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 Fmoc-Hyp(tBu)-OH (11.54 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) DMF (5 v/mmol, 70.3 ml)를 주입하여 용해한다. MC로 세척된 반응부에 DMF (4 v/mmol, 56.36 ml)주입 후 교반하면서 다른 반응부의 용해액을 주입한다. 주 반응부에 DIC (28.18 mmol, 4.4 ml)를 주입 후 상온에서 4 시간 교반하고 탈수하여 원하는 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Gly-Hyp(tBu)-Fmoc을 수득할 수 있다.20% piperidine (8 v/mmol) in Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Gly-Fmoc (14.09 mmol) synthesized in Example 9 , 113 ml) and stirred for 10 minutes after injection to dehydrate. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. Fmoc-Hyp(tBu)-OH (11.54 g, 28.18 mmol), HOBt (3.81 g, 28.18 mmol) and DMF (5 v/mmol, 70.3 ml) were injected into the other reaction part to dissolve. After DMF (4 v/mmol, 56.36 ml) was injected into the reaction part washed with MC, the solution of the other reaction part was injected while stirring. After injecting DIC (28.18 mmol, 4.4 ml) into the main reaction part, stir at room temperature for 4 hours and dehydrate to the desired Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt) )-Gly-Hyp(tBu)-Fmoc can be obtained.

실시예 11: Lys-Pro-Gly-Ala-Leu-Gly-Asp-Gln-Gly-Hyp의 제조Example 11: Preparation of Lys-Pro-Gly-Ala-Leu-Gly-Asp-Gln-Gly-Hyp

실시예 10에서 합성된 Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Gly-Hyp(tBu)-Fmoc (14.09 mmol)에 20 % piperidine (8 v/mmol, 113 ml) 주입 후 10 분 교반하여 탈수한다. 이 과정을 2 번 반복한다. MC (9 v/mmol, 127 mL) 주입 후 10 분 교반하여 탈수한다. 이 과정을 3 번 반복한다. 다른 반응부에 TFA (100 ml), MC (40 ml), H2O (1.4 ml)를 주입하여 4 시간 교반하여 반응액을 여과한다. 탈수된 고체를 MC (30 ml)로 세척 후 여과액에 합친 후 여과액 부피 1/2로 감압농축한다. 다른 반응부에 IPE (800 ml)를 주입한 후 농축액을 역적가하여 고체를 석출시킨다. 석출된 고체를 탈수하여 Lys-Pro-Gly-Ala-Leu-Gly-Asp-Gln-Gly-Hyp를 16 g 수득할 수 있다. (purity 71.43 %)20% piperidine in Trityl resin-Lys(Boc)-Pro-Gly-Ala-Leu-Gly-Asp(OtBu)-Gln(Trt)-Gly-Hyp(tBu)-Fmoc (14.09 mmol) synthesized in Example 10 (8 v/mmol, 113 ml) After injection, it is stirred for 10 minutes to dehydrate. Repeat this process 2 times. After injection of MC (9 v/mmol, 127 mL), it is stirred for 10 minutes to dehydrate. Repeat this process 3 times. TFA (100 ml), MC (40 ml), and H 2 O (1.4 ml) were injected into the other reaction part, stirred for 4 hours, and the reaction solution was filtered. The dehydrated solid is washed with MC (30 ml) and combined with the filtrate, and then concentrated under reduced pressure to 1/2 the volume of the filtrate. After injecting IPE (800 ml) into the other reaction part, the concentrate is added dropwise to precipitate a solid. By dehydrating the precipitated solid, 16 g of Lys-Pro-Gly-Ala-Leu-Gly-Asp-Gln-Gly-Hyp can be obtained. (purity 71.43%)

실시예 12: Lys-Pro-Gly-Ala-Leu-Gly-Asp-Gln-Gly-Hyp의 정제Example 12: Purification of Lys-Pro-Gly-Ala-Leu-Gly-Asp-Gln-Gly-Hyp

실시예 11에서 합성된 Lys-Pro-Gly-Ala-Leu-Gly-Asp-Gln-Gly-Hyp 5 g을 Prep LC를 통하여 정제한 후 감압농축을 통하여 완전농축하여 유기용매를 제거한 뒤, H2O로 용해하여 Prep LC를 통하여 이온교환수지를 이용하여 Acetate염으로 치환한다. 분액을 감압농축하여 IPA 20 ml을 주입하여 용해 후 IPE 120 ml를 주입하여 상온에서 교반한다. 석출된 고체는 탈수하여 IPE 10 ml로 세척 후 질소 및 진공건조를 통하여 최종 3.2 g을 수득하였다. (순도 99.8 %, 정제 수율: 64 %)Back after the thread time is it the Lys-Pro-Gly-Ala- Leu-Gly-Asp-Gln-Gly-Hyp 5 g prepared in 11 was purified via Prep LC by full concentration through concentration under reduced pressure to remove an organic solvent, with H2O Dissolve and replace with acetate salt using ion exchange resin through Prep LC. The liquid is concentrated under reduced pressure and dissolved by injecting 20 ml of IPA, then injecting 120 ml of IPE and stirring at room temperature. The precipitated solid was dehydrated, washed with 10 ml of IPE, and dried under nitrogen and vacuum to obtain a final 3.2 g. (purity 99.8%, purification yield: 64%)

상술한 실시예에 설명된 특징, 구조, 효과 등은 본 발명의 적어도 하나의 실시예에 포함되며, 반드시 하나의 실시예에만 한정되는 것은 아니다. 나아가, 각 실시예에서 예시된 특징, 구조, 효과 등은 실시예들이 속하는 분야의 통상의 지식을 가지는 자에 의하여 다른 실시예들에 대해서도 조합 또는 변형되어 실시 가능하다. 따라서 이러한 조합과 변형에 관계된 내용들은 본 발명의 범위에 포함되는 것으로 해석되어야 할 것이다. Features, structures, effects, etc. described in the above-described embodiments are included in at least one embodiment of the present invention, and are not necessarily limited to one embodiment. Furthermore, features, structures, effects, etc. illustrated in each embodiment can be combined or modified for other embodiments by those of ordinary skill in the art to which the embodiments belong. Accordingly, the contents related to such combinations and modifications should be interpreted as being included in the scope of the present invention.

또한, 이상에서 실시예들을 중심으로 설명하였으나 이는 단지 예시일 뿐 본 발명을 한정하는 것이 아니며, 본 발명이 속하는 분야의 통상의 지식을 가진 자라면 본 실시예의 본질적인 특성을 벗어나지 않는 범위에서 이상에 예시되지 않은 여러 가지의 변형과 응용이 가능함을 알 수 있을 것이다. 예를 들어, 실시예들에 구체적으로 나타난 각 구성 요소는 변형하여 실시할 수 있는 것이다. 그리고 이러한 변형과 응용에 관계된 차이점들은 첨부한 청구 범위에서 규정하는 본 발명의 범위에 포함되는 것으로 해석되어야 할 것이다. In addition, although the embodiments have been described above, these are merely examples and do not limit the present invention, and those of ordinary skill in the art to which the present invention pertains are exemplified above in a range that does not depart from the essential characteristics of the present embodiment. It can be seen that various modifications and applications that have not been made are possible. For example, each component specifically shown in the embodiments may be implemented by modification. And the differences related to these modifications and applications should be construed as being included in the scope of the present invention defined in the appended claims.

Claims (6)

트리틸기가 도입된 수지에 반응용매를 이용하여 아민말단 및 측쇄가 모두 보호된 아미노산 유도체들을 순차적으로 결합시키는 제1 단계; 및
상기 제1 단계의 수지에서 펩타이드를 분리하고 측쇄 보호기들을 제거하는 제2 단계의 공정을 수행함을 특징으로 하는 하기 구조식 (a)의 서열로 표시되는 펩타이드의 제조 방법.
구조식 (a) Hyp-Gly-Gln-Asp-Gly-Leu-Ala-Gly-Pro-Lys
A first step of sequentially binding amino acid derivatives in which both the amine terminus and the side chains are protected by using a reaction solvent to the trityl group-introduced resin; and
A method for producing a peptide represented by the sequence of the following structural formula (a), characterized in that performing the second step of separating the peptide from the resin of the first step and removing the side chain protecting groups.
Structural formula (a) Hyp-Gly-Gln-Asp-Gly-Leu-Ala-Gly-Pro-Lys
 제1항에 있어서,
상기 아미노산 유도체는 아민말단 및 측쇄가 Boc(tert-butoxycarbonyl), Fmoc(9-fluorenylmethoxycarbonyl), OtBu(tert-Butoxy), tBu(tert-Butyl) 및 Trt(Trityl)로 이루어진 군에서 선택된 어느 하나의 보호기로 보호되는 것을 특징으로 하는 제조 방법.
According to claim 1,
The amino acid derivative has any one protecting group selected from the group consisting of amine terminus and side chains Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), OtBu (tert-Butoxy), tBu (tert-Butyl) and Trt (Trityl). Manufacturing method, characterized in that protected with.
 제1항에 있어서,
상기 반응용매는 디클로메탄, 클로로포름, 디클로로에탄, 디메틸포름아마드, 디메틸아세트아미드, N-메틸피로리디논, 테트라히드로푸란, 트리플루오르아세트산, 디옥산 또는 이들의 혼합용매로부터 선택되는 것을 특징으로 하는 제조 방법.
According to claim 1,
The reaction solvent is selected from dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, N-methylpyrrolidinone, tetrahydrofuran, trifluoroacetic acid, dioxane, or a mixture thereof. manufacturing method.
 제1항에 있어서,
상기 펩타이드의 제조 방법은,
2-염화 클로로트리틸(Chlorotrityl chloride) 형 수지 를 준비하는 단계;
상기 수지에 반응용매로 Fmoc기를 제거하여 탈보호 반응을 수행하는 단계;
상기 반응물에 Fmoc-Pro, Fmoc-Gly, Fmoc-Ala, Fmoc-Leu, Fmoc-Gly, Fmoc-Asp(OtBu), Fmoc-Gln(Trt), Fmoc-Gly, 및 Fmoc-Hyp(tBu)을 순차적으로 첨가하여 커플링 반응시키는 단계;
상기 반응물의 Fmoc기를 탈보호하는 단계;
상기 반응물에서 수지를 제거하여 상기 구조식(a)의 서열로 표기되는 펩타이드를 수득하는 단계; 및
상기 펩타이드를 정제하는 단계를 포함하는 펩타이드 제조 방법.
According to claim 1,
The method for producing the peptide comprises:
Preparing 2-chlorotrityl chloride (Chlorotrityl chloride) type resin;
performing a deprotection reaction by removing the Fmoc group from the resin as a reaction solvent;
Fmoc-Pro, Fmoc-Gly, Fmoc-Ala, Fmoc-Leu, Fmoc-Gly, Fmoc-Asp(OtBu), Fmoc-Gln(Trt), Fmoc-Gly, and Fmoc-Hyp(tBu) were sequentially added to the reaction. adding to the coupling reaction;
deprotecting the Fmoc group of the reactant;
removing the resin from the reaction to obtain a peptide represented by the sequence of the structural formula (a); and
A method for producing a peptide comprising the step of purifying the peptide.
 제4항에 있어서,
상기 2-염화 클로로트리틸(chlorotrityl chloride) 형 수지에 Fmoc-Lys(Boc)를 붙인 2-염화 클로로트리틸 형 링크-라이신형 수지(link-Lys-Resin)의 링크는 하기 화학식 1의 링크 2-클로로트리틸 클로라이드(2- chlorotrityl chloride)인 제조 방법.

[화학식 1]
Figure pat00002

5. The method of claim 4,
The link of the 2-chlorotrityl type link-lysine type resin (link-Lys-Resin) in which Fmoc-Lys (Boc) is attached to the 2-chlorotrityl chloride type resin is Link 2 of Formula 1 below -Chlorotrityl chloride (2-chlorotrityl chloride) is a manufacturing method.

[Formula 1]
Figure pat00002

 제4항에 있어서,
상기 커플링 반응시키는 단계에서 사용되는 커플링 시약은 각 아미노산의 활성 에스테르, DCC(dicyclohexyl carbodiimide), DIC(diisopropyl carbodiimide), BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP(Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HBTU(O-Benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate), TBTU(O-(Benzotriaol-1-yl)- N,N,N',N'-tetramethyl-uronium tetrafluoroborate), HATU(2-(1H-7-Azabenzotriaol-1-yl)-1,1,3,3- tetramethyl uranium hexafluorophosphate Methanaminium), TATU(2-(1H-7-Azabenzotriaol-1-yl)- 1,1,3,3- tetramethyl uranium tetrafluoroborate Methanaminium) 및 CDI(carbonyl diimidazole)로 이루어진 군에서 선택된 적어도 어느 하나인 제조 방법.5. The method of claim 4,
The coupling reagent used in the coupling reaction step is an active ester of each amino acid, DCC (dicyclohexyl carbodiimide), DIC (diisopropyl carbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) , PyBOP(Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HBTU(O-Benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate), TBTU(O-(Benzotriaol-1-yl)-N,N, N',N'-tetramethyl-uronium tetrafluoroborate), HATU(2-(1H-7-Azabenzotriaol-1-yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate Methanaminium), TATU(2-(1H-7) -Azabenzotriaol-1-yl)- 1,1,3,3- tetramethyl uranium tetrafluoroborate Methanaminium) and CDI (carbonyl diimidazole) at least one method selected from the group consisting of.
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