KR20230050889A - Tripeptide containing histidine derivative and antibacterial composition containing the same - Google Patents
Tripeptide containing histidine derivative and antibacterial composition containing the same Download PDFInfo
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- KR20230050889A KR20230050889A KR1020210134217A KR20210134217A KR20230050889A KR 20230050889 A KR20230050889 A KR 20230050889A KR 1020210134217 A KR1020210134217 A KR 1020210134217A KR 20210134217 A KR20210134217 A KR 20210134217A KR 20230050889 A KR20230050889 A KR 20230050889A
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- South Korea
- Prior art keywords
- resin
- tfa
- formula
- tripeptide
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- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 30
- 150000002410 histidine derivatives Chemical class 0.000 title claims abstract description 27
- 239000000203 mixture Substances 0.000 title claims abstract description 24
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- 150000001413 amino acids Chemical class 0.000 claims description 14
- 125000006239 protecting group Chemical group 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 11
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- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 claims description 3
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- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000004184 methoxymethyl group Chemical group [H]C([H])([H])OC([H])([H])* 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000004092 methylthiomethyl group Chemical group [H]C([H])([H])SC([H])([H])* 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000004305 normal phase HPLC Methods 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 125000006503 p-nitrobenzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1[N+]([O-])=O)C([H])([H])* 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- ATLPLEZDTSBZQG-UHFFFAOYSA-N propan-2-ylphosphonic acid Chemical compound CC(C)P(O)(O)=O ATLPLEZDTSBZQG-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001981 tert-butyldimethylsilyl group Chemical group [H]C([H])([H])[Si]([H])(C([H])([H])[H])[*]C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- MBYLVOKEDDQJDY-UHFFFAOYSA-N tris(2-aminoethyl)amine Chemical compound NCCN(CCN)CCN MBYLVOKEDDQJDY-UHFFFAOYSA-N 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
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Abstract
Description
본 발명은 히스티딘 유도체를 포함하는 트라이펩타이드 및 이를 포함하는 항균 조성물에 관한 것으로서, 더욱 상세하게는 비브리오균에 대하여 항균활성을 갖는 히스티딘 유도체를 포함하는 트라이펩타이드 및 이를 포함하는 항균 조성물에 관한 것이다.The present invention relates to a tripeptide containing a histidine derivative and an antibacterial composition containing the same, and more particularly, to a tripeptide containing a histidine derivative having antibacterial activity against Vibrio and an antibacterial composition containing the same.
펩타이드는 바이오의 핵심소재로 단백질의 기능적 최소 단위이며, 아미노산이 2개로부터 50개 이하로 구성된다. 소량으로 우수한 효능을 보일 뿐만 아니라 독성이 없어 의약품, 식품 및 화장품의 주요 원료로 많이 활용되고 있다. 특히 인체에 안전하고 효과가 좋은 펩타이드 화장품 소재는 날로 사용량이 늘어날 뿐만 아니라 새로운 소재의 개발은 수입대체 효과 등 당 산업에서 매우 중요하다고 할 수 있다.Peptide is a core material of bio and is the smallest functional unit of protein, and consists of 2 to 50 amino acids. Not only does it show excellent efficacy in small amounts, but it is also widely used as a major raw material for medicines, foods, and cosmetics because it is non-toxic. In particular, the use of peptide cosmetic materials that are safe and effective for the human body is increasing day by day, and the development of new materials is very important in the industry, such as import substitution effect.
또한 펩티도미메틱 (Peptidomimetic)은 펩타이드의 유사체로서, 펩타이드의 장점인 높은 효능은 그대로 유지시키면서 단점인 체내 안정성을 획기적으로 극복시킬수 있는 핵심 차세대 바이오 소재이다.In addition, Peptidomimetic is an analog of peptide, and is a key next-generation biomaterial that can dramatically overcome the in vivo stability, which is a disadvantage of peptides, while maintaining high efficacy, which is an advantage of peptides.
2000년 이후 합성 기술의 발달로 그동안 상업화가 이루어지지 않았던 펩타이드의 상업화 및 연구가 본격적으로 이루어지기 시작하였으며, 이를 바탕으로 한 신약 연구 또한 매년 꾸준히 증가하고 있는 추세이다.Since 2000, with the development of synthetic technology, commercialization and research on peptides, which had not been commercialized until now, began to be conducted in earnest, and research on new drugs based on this has also steadily increased every year.
한편, 비브리오균 (Vibrio parahaemolyticus)은 바닷물에서 서식하는 식중독균으로 연안 해역의 바닷물, 갯벌, 어패류에서 주로 검출되며, 여름철에 따뜻한 바닷물에서 증식한 장염 비브리오균이 생선, 조개, 오징어 등의 표피, 아가미, 내장 등에 부착하여 이를 섭취한 사람에게 식중독을 일으킨다. 일반적으로 비브리오균은 바닷물 온도가 15℃ 이상이 되면 증식을 시작하며, 20~37℃에서 3~4시간 만에 100만 배로 증가한다. 비브리오균은 여름철 어패류에 서식하므로, 이를 날것으로 먹었을 경우 감염되어 매우 짧은 기간내에 폐혈증이 유발되고, 사망에 이르게 되지만, 이를 치료하기 위한 약품의 개발은 미진한 실정이다. On the other hand, Vibrio parahaemolyticus is a food poisoning bacterium that lives in seawater and is mainly detected in seawater, tidal flats, and fish and shellfish in coastal waters. It attaches to the intestines and causes food poisoning to those who ingest it. In general, Vibrio begins to proliferate when the seawater temperature is higher than 15 ° C, and increases to 1 million times in 3 to 4 hours at 20 ~ 37 ° C. Vibrio bacteria live in fish and shellfish in the summer, so when eaten raw, they become infected and cause sepsis and death within a very short period of time, but the development of drugs to treat this is insufficient.
패혈증을 일으키는 비브리오균은 '비브리오 불니피쿠스'이며, 비브리오 패혈증은 오한, 발열 등의 신체 전반에 걸친 증상과 설사, 복통, 하지 통증 등을 유발한다. 비브리오 패혈증은 만성 간질환 등의 만성 질환을 앓고 있어 면역기능이 떨어진 사람에게서 잘 감염되며, 어패류를 날 것이나 덜 익힌 채로 먹었을 때나, 어패류·바닷물·갯벌에 있는 비브리오 불니피쿠스균이 피부 상처에 접촉되었을 때 감염된다.The Vibrio bacteria that causes sepsis is 'Vibrio vulnificus', and Vibrio sepsis causes symptoms throughout the body, such as chills and fever, as well as diarrhea, abdominal pain, and lower extremity pain. Vibrio sepsis is easily infected by people with weakened immune function due to chronic diseases such as chronic liver disease, and when fish and shellfish are eaten raw or undercooked, or when Vibrio bulnificus bacteria in fish and shellfish, seawater, and tidal flats come into contact with skin wounds. become infected when
한국등록특허 제2037383호는 비브리오균에 대한 항균활성을 갖는 박테리오파지 및 이를 포함하는 항생용 조성물을 개시하였고, 한국등록특허 제1151878호는 지방산 트리펩타이드염 및 이를 함유하는 항균 조성물을 개시하였으나, 비브리오균에 우수한 항균활성을 갖는 펩타이드에 대한 연구는 많지 않았다. Korean Patent No. 2037383 discloses a bacteriophage having antibacterial activity against Vibrio and an antibiotic composition containing the same, and Korean Patent No. 1151878 discloses a fatty acid tripeptide salt and an antibacterial composition containing the same. There have not been many studies on peptides with excellent antibacterial activity.
이에, 본 발명자들은 위에서 언급한 바와 같이 히스티딘 유도체를 포함하는 다양한 항균 펩티도미메틱을 연구, 개발하는 과정에서 특정 서열을 갖는 펩티도미메틱인 히스티딘 유도체를 포함하는 트라이펩타이드가 비브리오균에 우수한 항균 활성을 갖는다는 것을 확인하고, 본 발명을 완성하게 되었다.Therefore, as mentioned above, the present inventors, in the process of researching and developing various antibacterial peptidomimetics containing histidine derivatives, found that tripeptides containing histidine derivatives, which are peptidomimetics having specific sequences, have excellent antibacterial activity against Vibrio bacteria. It was confirmed that it has, and the present invention was completed.
본 발명의 목적은 비브리오균에 특이적으로 작용하는 히스티딘 유도체를 포함하는 트라이펩타이드 및 이를 포함하는 항균 조성물을 제공하는데 있다.An object of the present invention is to provide a tripeptide containing a histidine derivative that acts specifically on Vibrio bacteria and an antibacterial composition containing the tripeptide.
본 발명의 다른 목적은 비브리오균에 특이적으로 작용하는 히스티딘 유도체를 포함하는 트라이펩타이드의 제조방법을 제공하는데 있다.Another object of the present invention is to provide a method for producing a tripeptide containing a histidine derivative that acts specifically on Vibrio bacteria.
상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 I의 구조를 갖는 히스티딘 유도체를 포함하는 트라이펩타이드를 제공한다.In order to achieve the above object, the present invention provides a tripeptide containing a histidine derivative having the structure of Formula I below.
[화학식 I] [Formula I]
본 발명은 또한, 상기 화학식 I의 구조를 갖는 히스티딘 유도체를 포함하는 트라이펩타이드를 유효성분으로 함유하는 항균용 조성물을 제공한다.The present invention also provides an antibacterial composition containing, as an active ingredient, a tripeptide containing a histidine derivative having the structure of Formula I.
본 발명에 있어서, 상기 항균용 조성물은 비브리오균에 대하여 항균활성을 갖는 것을 특징으로 한다. In the present invention, the antimicrobial composition is characterized in that it has an antibacterial activity against Vibrio bacteria.
본 발명은 또한, (a) 고체상 (Solid-phase) 합성방법으로 화학식 1로 표시되는 Fmoc-His(mono-cyclohexylpropyl)-NH-레진을 수득하는 단계; (b) 상기 단계 (a)에서 수득한 레진에 Fmoc-His(mono-cyclohexylpropyl)-OH 및 Fmoc-Arg(보호기)-OH를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득하는 단계; (c) 상기 단계 (b)에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 트라이펩타이드를 수득하는 단계; 및 (d) 상기 단계 (c)에서 수득한 비정제 트라이펩타이드를 정제하여 화학식 I로 표시되는 트라이펩타이드를 수득하는 단계로 구성된 히스티딘 유도체를 포함하는 트라이펩타이드의 제조방법을 제공한다.The present invention also, (a) obtaining Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by
[화학식 1][Formula 1]
[화학식 2] [Formula 2]
[화학식 3][Formula 3]
[화학식 I] [Formula I]
본 발명에 있어서, 상기 레진은 링크아마이드 레진 (Rink Amide Resin), 링크아마이드 엠비에이치에이 레진 (Rink Amide MBHA Resin), 링크아마이드 에이엠 레진 (Rink Amide AM Resin) 및 링크아마이드 렘 레진 (Rink Amide RAM Resin)으로 구성된 군에서 선택되는 것을 특징으로 한다.In the present invention, the resins are Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin. ) It is characterized in that selected from the group consisting of.
본 발명에 있어서, 레진 및 아미노산의 보호기를 동시에 제거하는 산성 용액은 TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95/5), TFA/DCM/indole (70/28/2) 및 TFA/TIPS/water (95/2.5/2.5)로 구성된 군에서 선택되는 것을 특징으로 한다.In the present invention, the acidic solution that simultaneously removes the protecting group of the resin and amino acid is TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/ 5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA/phenol (95 /5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95/ 5), TFA/DCM/indole (70/28/2) and TFA/TIPS/water (95/2.5/2.5).
본 발명의 히스티딘 유도체를 포함하는 트라이펩타이드는 그람 음성균, 특히 비브리오균에 대한 항균 효과가 우수하며, 인체 적용시 체내 안정성이 우수하므로, 의약품 등의 산업 분야에 매우 유용하다. The tripeptide containing the histidine derivative of the present invention has an excellent antibacterial effect against Gram-negative bacteria, particularly Vibrio bacteria, and excellent stability in the body when applied to the human body, so it is very useful in industrial fields such as pharmaceuticals.
도 1은 본 발명의 일 실시예에 따른 히스티딘 유도체를 포함하는 트라이펩타이드의 합성 공정도이다.
도 2는 본 발명에 따른 히스티딘 유도체를 포함하는 트라이펩타이드의 분자량 측정 (MALDI-TOF MS) 결과이다.
도 3은 본 발명에 따른 히스티딘 유도체를 포함하는 트라이펩타이드의 순도를 측정한 HPLC Chromatogram 결과이다.
도 4는 본 발명에 따른 히스티딘 유도체를 포함하는 트라이펩타이드의 비브리오균에 대한 항균 활성을 평가한 Disc Diffusion assay 사진이다.
도 5는 본 발명에 따른 히스티딘 유도체를 포함하는 트라이펩타이드의 비브리오균에 대한 성장 억제 효과를 평가한 Alamar blue assay 결과이다.1 is a process diagram for synthesizing a tripeptide including a histidine derivative according to an embodiment of the present invention.
2 is a result of molecular weight measurement (MALDI-TOF MS) of a tripeptide containing a histidine derivative according to the present invention.
3 is an HPLC chromatogram result of measuring the purity of a tripeptide containing a histidine derivative according to the present invention.
4 is a picture of a Disc Diffusion assay evaluating the antibacterial activity of the tripeptide containing histidine derivatives against Vibrio according to the present invention.
5 is an Alamar blue assay result of evaluating the growth inhibitory effect of the tripeptide containing a histidine derivative according to the present invention on Vibrio bacteria.
본 명세서에서 특별한 표시가 없는 한, 아미노산 및 보호기의 지정에 사용되는 약어는 IUPAC-IUB의 생화학 용어 위원회 (Commission of Biochemical Nomenclature)에서 권장하는 용어에 기초한다 (Biochemistry, 11:1726-1732(1972); Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).Unless otherwise indicated herein, the abbreviations used for the designation of amino acids and protecting groups are based on terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Biochemistry, 11:1726-1732(1972)). ( Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).
본 명세서에서 용어 "펩타이드"는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미한다.As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by a peptide bond.
본 명세서에서 사용한 용매, 시약, 보호기 및 아미노산의 약어는 다음과 같다:Abbreviations for solvents, reagents, protecting groups and amino acids used herein are as follows:
DCM: 디클로로메탄 (Dichloromethane)DCM: Dichloromethane
DMSO: 디메틸설폭시드 (Dimethylsulfoxide)DMSO: Dimethylsulfoxide
DTT: 디티올쓰레이톨 (Dithiolthreitol)DTT: Dithiolthreitol
EDT: 1,2-에탄디티올 (1,2-Ethanedithiol)EDT: 1,2-Ethanedithiol
ACN: 아세토니트릴 (Acetonitrile)ACN: Acetonitrile
Arg: 아르기닌 (Arginine)Arg: Arginine
His: 히스티딘 (Histidine)His: Histidine
HPLC: 고성능액체크로마토그래피 (High Performance Liquid Chromatography)HPLC: High Performance Liquid Chromatography
Fmoc: 9-플루오레닐옥시카보닐 (9-Fluorenyloxycarbonyl)Fmoc: 9-Fluorenyloxycarbonyl
Trt: 트리페닐메틸 (또는 트리틸) (Triphenylmethyl or Trityl)Trt: Triphenylmethyl or Trityl
Pbf: 2,2,4,6,7-펜타메틸-디히드로벤조퓨란-5-설포닐 (2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl)Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl)
Pmc: 2,2,5,7,8-펜타메틸크로만-6-설포닐 (2,2,5,7,8-Pentamethylchroman-6-sulfonyl)PMC: 2,2,5,7,8-pentamethylchroman-6-sulfonyl (2,2,5,7,8-Pentamethylchroman-6-sulfonyl)
Mtr: 4-메톡시-2,3,6-트리메틸페닐-설포닐 (4-Methoxy-2,3,6-trimethylphenyl-sulfonyl)Mtr: 4-methoxy-2,3,6-trimethylphenyl-sulfonyl (4-Methoxy-2,3,6-trimethylphenyl-sulfonyl)
Tos: 파라-톨루엔설포닐 (para-Toluenesulfonyl)Tos: para-Toluenesulfonyl
TES: 트라이에틸실란 (Triethylsilane)TES: Triethylsilane
TFA: 트라이플루오로아세틱 엑시드 (Trifluoroacetic acid)TFA: Trifluoroacetic acid
TIPS: 트리이소프로필실란 (Triisopropylsilane)TIPS: Triisopropylsilane
본 발명에서는 히스티딘 유도체를 포함하는 다양한 항균 펩티도미메틱을 연구, 개발하는 과정에서 특정 서열을 갖는 펩티도미메틱인 히스티딘 유도체를 포함하는 트라이펩타이드를 제조하였고, 이는 그람 음성균, 특히 비브리오균에 대하여 우수한 항균 활성을 갖는다는 것을 확인하였다.In the present invention, a tripeptide containing a histidine derivative, which is a peptidomimetic having a specific sequence, was prepared in the course of research and development of various antibacterial peptidomimetics including histidine derivatives. It was confirmed that it has activity.
따라서, 본 발명은 일 관점에서 하기 화학식 I의 구조를 갖는 히스티딘 유도체를 포함하는 트라이펩타이드에 관한 것이다. Accordingly, in one aspect, the present invention relates to a tripeptide comprising a histidine derivative having the structure of Formula I below.
[화학식 I] [Formula I]
본 발명은 또한, 상기 화학식 I의 구조를 갖는 히스티딘 유도체를 포함하는 트라이펩타이드를 유효성분으로 함유하는 항균용 조성물에 관한 것이다.The present invention also relates to an antibacterial composition containing, as an active ingredient, a tripeptide containing a histidine derivative having the structure of Formula I.
상기 항균용 조성물은 그람 음성균에 대한 항균활성을 갖고 있으며, 그람 음성균은 비브리오 spp., 살모넬라 spp. 등을 예시할 수 있으나 이에 한정되는 것은 아니다. 상기 비브리오 균은 비브리오 불니피쿠스 (Vibrio vulnificus), 비브리오 알기놀리티쿠스 (Vibrio alginolyticus), 비브리오 파라해모리티쿠스 (Vibrio parahaemolyticus) 등을 예시할 수 있다.The antimicrobial composition has antibacterial activity against Gram-negative bacteria, and Gram-negative bacteria are Vibrio spp., Salmonella spp. etc. can be exemplified, but is not limited thereto. Examples of the Vibrio bacteria include Vibrio vulnificus , Vibrio alginolyticus , and Vibrio parahaemolyticus .
본 발명의 항균용 조성물이 약학적 조성물로 사용하는 경우, 피부, 주사, 피하주사, 복용제제 등에 사용할 수 있다. When the antibacterial composition of the present invention is used as a pharmaceutical composition, it can be used for skin, injection, subcutaneous injection, oral preparation, and the like.
본 발명의 항균용 조성물에 포함되는 화학식 I의 구조를 갖는 히스티딘 유도체를 포함하는 트라이펩타이드의 함량은 용도, 적용 형태, 사용 목적 및 소망하는 효과에 따라서 적절히 조절 가능하며, 함량 대비 효과를 고려하여, 예컨대 전체 조성물 중량에 대하여 0.0001 내지 99.9 중량% 이내에서 사용할 수 있다.The content of the tripeptide containing the histidine derivative having the structure of formula (I) included in the antibacterial composition of the present invention can be appropriately adjusted according to the use, application form, purpose of use and desired effect, and considering the effect of the content, For example, it may be used within 0.0001 to 99.9% by weight based on the total weight of the composition.
본 발명의 항균용 조성물은 경구 및 비경구로 투여할 수 있으며, 경구, 경피, 피하, 정맥투여가 가능하며, 또한 경피 부여와 도포에 의한 국부 투여 (Topical application) 방식으로 적용될 수 있다.The antibacterial composition of the present invention can be administered orally and parenterally, and can be administered orally, transdermally, subcutaneously, and intravenously, and can also be applied in a topical application method by applying and applying transdermally.
본 발명은 또한, (a) 고체상 (Solid-phase) 합성방법으로 화학식 1로 표시되는 Fmoc-His(mono-cyclohexylpropyl)-NH-레진을 수득하는 단계; (b) 상기 단계 (a)에서 수득한 레진에 Fmoc-His(mono-cyclohexylpropyl)-OH 및 Fmoc-Arg(보호기)-OH를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득하는 단계; (c) 상기 단계 (b)에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 트라이펩타이드를 수득하는 단계; 및 (d) 상기 단계 (c)에서 수득한 비정제 트라이펩타이드를 정제하여 화학식 I로 표시되는 트라이펩타이드를 수득하는 단계로 구성된 히스티딘 유도체를 포함하는 트라이펩타이드의 제조방법에 관한 것이다.The present invention also, (a) obtaining Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by
[화학식 1][Formula 1]
[화학식 2] [Formula 2]
[화학식 3][Formula 3]
[화학식 I] [Formula I]
이하 각 단계별로 본 발명의 히스티딘 유도체를 포함하는 트라이펩타이드의 제조방법을 설명한다. Hereinafter, the method for preparing the tripeptide containing the histidine derivative of the present invention will be described in each step.
(a) 단계: 고체상 (Solid-phase) 합성방법으로 화학식 1로 표시되는 Fmoc-His(mono-cyclohexylpropyl)-NH-레진을 수득한다.Step (a): Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by
상기 레진으로는 링크아마이드 레진 (Rink Amide Resin), 링크아마이드엠비에이치에이 레진 (Rink Amide MBHA Resin), 링크아마이드에이엠 레진 (Rink Amide AM Resin), 링크아마이드렘 레진 (Rink Amide RAM Resin) 등을 이용할 수 있다.As the resin, Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, Rink Amide RAM Resin, etc. can be used. can
본 발명에 있어서 사용되는 용매로는 디클로로메탄 (Dichloromethane), N,N-디메틸포름아마이드 (N,N-Dimethylformamide), N,N-디메틸아세트아마이드 (N,N-Dimethylacetamide), N-메틸피롤리돈 (N-Methylpyrrolidone), 클로로포름 (Chloroform), 1,2-디클로로에탄 (1,2-Dichloroethane), 테트라히드로퓨란 (Tetrahydrofurane), 1,4-디옥산 (1,4-Dioxane), 메탄올 (Methanol), 에탄올 (Ethanol), 이소프로판올 (Isopropanol), 에틸렌 글리콜 (Ethylene glycol), 메틸 아세테이트 (Methyl acetate), 에틸 아세테이트 (Ethyl acetate) 등을 예시할 수 있으나 이에 한정되는 것은 아니다. Solvents used in the present invention include dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrroly N-Methylpyrrolidone, Chloroform, 1,2-Dichloroethane, Tetrahydrofurane, 1,4-Dioxane, Methanol ), ethanol, isopropanol, ethylene glycol, methyl acetate, ethyl acetate, and the like, but are not limited thereto.
(b) 단계: (a) 단계에서 수득한 레진에 Fmoc-His(mono-cyclohexylpropyl)-OH 및 Fmoc-Arg(보호기)-OH를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득한다.Step (b): Fmoc-His (mono-cyclohexylpropyl) -OH and Fmoc-Arg (protecting group) -OH are sequentially bonded to the resin obtained in step (a) to obtain a protected peptide represented by
상기 (b) 단계는 다시 (ⅰ) 레진에 결합되어 있는 아미노산 보호기 (N-말단의 아미노기)를 염기로 처리하여 제거하고 레진을 세척하는 단계; (ⅱ) 새롭게 결합시킬 보호화된 아미노산을 세척된 레진에 반응 및 결합시키는 단계를 포함한다.The step (b) may further include (i) removing the amino acid protecting group (N-terminal amino group) bound to the resin by treating it with a base and washing the resin; (ii) reacting and binding the newly bound protected amino acid to the washed resin.
본 발명의 화학식 2에서 보호기는 메톡시메틸 (Methoxymethyl)기, 벤질옥시메틸 (Benzyloxymethyl)기, 트리페닐메틸 (Triphenylmethyl)기, 터트-부틸디메틸실릴 (tert-Butyldimethylsilyl)기, 터트-부틸디페닐실릴 (tert-Butyldiphenylsilyl)기,페닐디메틸실릴 (Phenyldimethylsilyl)기, 트리페닐실릴 (Triphenylsilyl)기, 트리에틸실릴 (Triethylsilyl)기, 트리이소프로필실릴 (Triisopropylsilyl)기, 파라-메톡시벤질 (para-Methyloxybenzyl)기, 테트라히드로피란 (Tetrahydropyran)기, 테트라히드로퓨란 (Tetrahydrofuran)기, 터트-부틸 (tert-Butyl)기, 디페닐메틸 (Diphenylmethyl)기, 2-클로로트리틸 (2-Chlorotrityl)기, 벤질 (Benzyl)기, o-니트로벤질(o-Nitrobenzyl)기, p-니트로벤질 (para-Nitrobenzyl)기, 9-페닐잔틸 (9-Phenylxanthyl)기, 메톡시트리틸 (Methoxytrityl)기, 알릴 (Allyl)기, 메틸 (Methyl)기, 메톡시에톡시메틸 (Methoxyethoxymethyl)기, 에톡시에틸 (Ethoxyethyl)기, 메틸티오메틸 (Methylthiomethyl)기, 2-나프틸메틸 (2-Naphtylmethyl)기, 터트-부틸옥시카르보닐 (tert-Butyloxycarbonyl)기, 벤질옥시카르보닐 (Benzyloxycarbonyl), 아세틸 (Acetyl)기, 벤조일 (Benzoyl)기, 파라-톨루엔술포닐 (para-Toluenesulfonyl)기, 2,4,6-트라이메틸페닐술포닐 (2,4,6-Trimethylphenylsulfonyl, mesityl-2-sulfonyl)기, 4-메톡시-2,3,6-트라이메틸페닐술포닐 (4-Methoxy-2,3,6-trimethylphenylsulfonyl)기, Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl)기, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)기, MIS (1,2-dimethylindole-3-sulfonyl)기, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl)기, NO₂(Nitro)기 등을 예시할 수 있으며, 파라-톨루엔술포닐 (para-Toluenesulfonyl)기, 2,4,6-트라이메틸페닐술포닐 (2,4,6-Trimethylphenylsulfonyl, mesityl-2-sulfonyl)기, 4-메톡시-2,3,6-트라이메틸페닐술포닐 (4-methoxy-2,3,6-trimethylphenylsulfonyl)기, Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl)기, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)기, MIS (1,2-dimethylindole-3-sulfonyl)기, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl)기, NO₂(Nitro)기를 이용하는 것이 바람직하며, Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl)기, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)기, MIS (1,2-dimethylindole-3-sulfonyl)기, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl)기, NO₂(Nitro)기를 이용하는 것이 보다 바람직하며, 아미드 보호기가 Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)를 이용하는 것이 가장 바람직하지만 이에 국한되는 것은 아니다.In Formula 2 of the present invention, the protecting group is a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group, a tert -butyldimethylsilyl group, a tert-butyldiphenylsilyl group ( tert -Butyldiphenylsilyl) group, phenyldimethylsilyl group, triphenylsilyl group, triethylsilyl group, triisopropylsilyl group, para-methoxybenzyl ( para -Methyloxybenzyl) group, tetrahydropyran group, tetrahydrofuran group, tert -butyl group, diphenylmethyl group, 2-chlorotrityl group, benzyl ( Benzyl) group , o-Nitrobenzyl group, p-nitrobenzyl group, 9-phenylxanthyl group, methoxytrityl group, allyl group, methyl group, methoxyethoxymethyl group, ethoxyethyl group, methylthiomethyl group, 2-naphtylmethyl group, tert-butyloxy Carbonyl ( tert -Butyloxycarbonyl) group, benzyloxycarbonyl (Benzyloxycarbonyl), acetyl (Acetyl) group, benzoyl (Benzoyl) group, para-toluenesulfonyl ( para -Toluenesulfonyl) group, 2,4,6-trimethylphenyl group phonyl (2,4,6-Trimethylphenylsulfonyl, mesityl-2-sulfonyl) group, 4-methoxy-2,3,6-trimethylphenylsulfonyl (4-Methoxy-2,3,6-trimethylphenylsulfonyl) group, Pmc ( 2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS (1,2-dimethylindole-3-sulfonyl) Group, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, NO₂ (Nitro) group, etc. can be exemplified, and para-Toluenesulfonyl ( para -Toluenesulfonyl) group, 2,4,6-trimethylphenyl Sulfonyl (2,4,6-Trimethylphenylsulfonyl, mesityl-2-sulfonyl) group, 4-methoxy-2,3,6-trimethylphenylsulfonyl (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS (1,2-dimethylindole-3-sulfonyl) group ) group, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, NO₂ (Nitro) group is preferred, and Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS (1,2-dimethylindole-3-sulfonyl) group, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, NO₂ It is more preferable to use a (Nitro) group, and it is most preferable to use Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) as the amide protecting group, but is not limited thereto.
상기 아미노산 보호기 (N-말단의 아미노기)를 제거하는 염기로는 피페리딘 (Piperidine), 4-메틸피페리딘 (4-Methylpiperidine), 피롤리딘 (Pyrrolidine), 피페라진 (Piperazine), 하이드라진 하이드레이트 (Hydrazine hydrate), DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene), 4-메틸피페리딘 (4-Methylpiperidine), 1-메틸-3-부틸이미다졸리윰 테트라플로오로보란 (1-methyl-3-butyl imidazolium BF4), 에탄올아민 (Ethanolamine), 시클로헥실아민 (Cyclohexylamine), 디시클로헥실아민 (Dicyclohexylamine), 트리스(2-아미노에틸)아민 (Tris(2-aminoethyl)amine), 1,3-디시클로헥산비스-(메틸아민) (1,3-Dicyclohexanebis-(methylamine)), 1,4-비스-(3-아미노프로필)피페라진 (1,4-Bis-(3-aminopropyl)piperazine), 디에틸아민 (Diethylamine), 4-디메틸아미노피리딘 (4-Dimethylaminopyridine) 등의 유기 염기 또는 수산화 리튬 (Lithium hydroxide), 수산화 나트륨 (Sodium Hydroxide), 수산화 칼슘 (Calcium Hydroxide), 수산화 칼륨 (Potassium Hydroxide) 등의 무기 염기를 이용할 수 있다.Bases that remove the amino acid protecting group (N-terminal amino group) include piperidine, 4-methylpiperidine, pyrrolidine, piperazine, hydrazine hydrate (Hydrazine hydrate), DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene), 4-Methylpiperidine, 1-methyl-3-butylimidazolium tetrafluoro Borane (1-methyl-3-butyl imidazolium BF4), Ethanolamine, Cyclohexylamine, Dicyclohexylamine, Tris(2-aminoethyl)amine ), 1,3-Dicyclohexanebis-(methylamine) (1,3-Dicyclohexanebis-(methylamine)), 1,4-bis-(3-aminopropyl)piperazine (1,4-Bis-(3 -aminopropyl)piperazine), diethylamine, 4-dimethylaminopyridine, or organic bases such as Lithium hydroxide, Sodium Hydroxide, Calcium Hydroxide, Hydroxide Inorganic bases such as potassium (Potassium Hydroxide) may be used.
본 발명에 있어서, 보호화된 아미노산을 세척된 레진에 반응 및 결합시킬 때 사용하는 시약으로는 DCC (N,N-Dicyclohexylcarbodiimide), DIC (N,N-Diisopropylcarbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), PyBrOP (Bromo-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate), PyOxim (Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), HCTU (2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N',N',-tetramethylaminium hexafluorophosphate), HDMC (N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uronium hexafluorophosphate N-oxide), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminiu m), COMU (1-[1-(Cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate), TOTT (2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate), EDCㅇHCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), TFFH (Tetramethylfluoroformamidinium hexafluorophosphate), EEDQ (N-Ethoxycarbony-2-ethoxy-1,2-dihydro-quinoline), T3P (2-Propanephosphonic acid anhydride), DEPBT (3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), HOOBt(HODhbt, Hydroxy-3,4-dihydro-4-ox-1,2,3-benzo-triazine), BTC (bis-Trichloromethylcarbonate 또는 Triphosgene), CDI (1,1'-Carbonyldiimidazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), HOSu (N-Hydroxysuccinimide) 등을 예시할 수 있다. In the present invention, reagents used when reacting and binding protected amino acids to the washed resin include DCC (N,N-Dicyclohexylcarbodiimide), DIC (N,N-Diisopropylcarbodiimide), BOP (Benzotriazole-1-yl- oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), PyBrOP (Bromo-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate) , PyOxim (Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), HCTU (2-(6 -Chloro-1H-benzotriazol-1-yl)-N,N,N',N',-tetramethylaminium hexafluorophosphate), HDMC (N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino] -uronium hexafluorophosphate N-oxide), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1 ,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), COMU (1-[1-(Cyano-2- ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate), TOTT (2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate), EDCㅇ HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), TFFH (Tetramethylfluoroformamidinium hexafluorophosphate), EEDQ (N-Ethoxycarbony-2-ethoxy-1,2-dihydro-quinoline), T3P (2-propanephosphonic acid anhydride) , DEPBT (3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), HOOBt (HODhbt, Hydroxy-3,4-dihydro- 4-ox-1,2,3-benzo-triazine), BTC (bis-Trichloromethylcarbonate or Triphosgene), CDI (1,1'-Carbonyldiimidazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), HOSu (N-Hydroxysuccinimide), and the like can be exemplified.
(c) 단계: (b) 단계에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 트라이펩타이드를 수득한다. Step (c): From the resin to which the protected peptide obtained in step (b) is bound, the protecting groups of the resin and the amino acid are simultaneously removed to obtain a non-purified tripeptide represented by
상기 레진 및 아미노산의 보호기를 동시에 제거하는 과정은 산성 용액의 존재 하에서 수행되는데, 상기 산성 용액은 TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95/5), TFA/DCM/indole (70/28/2) 및 TFA/TIPS/water (95/2.5/2.5) 등의 용액을 예시할 수 있다.The process of simultaneously removing the protecting groups of the resin and the amino acid is performed in the presence of an acidic solution, which is TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2 ), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5) , TFA/water (95/5), TFA/DCM/indole (70/28/2) and TFA/TIPS/water (95/2.5/2.5) solutions.
(d) 단계: (c) 단계에서 수득한 비정제 트라이펩타이드를 정제하여 화학식 I로 표시되는 트라이펩타이드를 수득한다.Step (d): The tripeptide represented by Formula I is obtained by purifying the crude tripeptide obtained in step (c).
상기 정제는 펩타이드 정제에 통상적으로 이용되는 방법으로 정제 가능하며, 순상 또는 역상 고성능액체크로마토그래피 (HPLC)를 이용할 수 있다. 정제시 사용되는 용매로는 메탄올 (Methanol), 에탄올 (Ethanol), 이소프로판올 (Isopropanol), 아세토니트릴 (Acetonitrile), 정제수 및 이들의 혼합용액으로 구성된 군에서 선택될 수 있으며, 삼불화아세트산 (Trifluoroacetic acid), 아세트산 (Acetic acid), 포름산 (Formic acid) 등의 산을 함께 이용할 수 있다.The purification may be performed by a method commonly used for peptide purification, and normal phase or reverse phase high performance liquid chromatography (HPLC) may be used. The solvent used for purification may be selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, purified water, and a mixture thereof, and trifluoroacetic acid , acids such as acetic acid and formic acid may be used together.
이하, 본 발명을 실시 예에 의해 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시 예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
[실시예][Example]
본 명세서 전체에 거쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 한 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (weight/volume) % for solid/solid, and liquid /liquid is (volume/volume) %.
실시 예 1 : H-His(mono-cyclohexylpropyl)-NH-레진 (Rink Amide)의 제조Example 1: Preparation of H-His (mono-cyclohexylpropyl) -NH-resin (Rink Amide)
여과막이 장착된 고체상 (solid-phase) 합성 반응기에 링크아마이드 에이엠 레진 (치환율 = 0.70 mmol/g, 100 mmole) 및 N,N-디메틸포름아마이드 (1000 ml, 이하 DMF)를 넣고, 10분간 레진을 팽창시킨 후, 감압 하에서 여과막을 통하여 용매를 제거하였다. Fmoc-His(mono-cyclohexylpropyl)-OH (분자량 = 501.62 g/mol) (150.49 g, 300 mmol, 3.0 당량), Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 5시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반한 다음, 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-His(mono-cyclohexylpropyl)-NH-레진(Rink Amide)을 수득하였다 (치환율: 0.7 mmol/g, 수율 >99%).Linkamide AM resin (substitution rate = 0.70 mmol/g, 100 mmole) and N,N-dimethylformamide (1000 ml, hereinafter referred to as DMF) were put in a solid-phase synthesis reactor equipped with a filtration membrane, and the resin was allowed to boil for 10 minutes. After swelling, the solvent was removed through a filtration membrane under reduced pressure. Fmoc-His(mono-cyclohexylpropyl)-OH (molecular weight = 501.62 g/mol) (150.49 g, 300 mmol, 3.0 equiv) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF. then put in. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution, and the mixture was slowly stirred at room temperature for 5 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of a 20% piperidine/DMF solution was added and stirred for 15 minutes, then the reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was obtained (substitution rate: 0.7 mmol/g, yield >99%).
실시 예 2 : H-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-레진 (Rink Amide)의 제조Example 2: Preparation of H-His (mono-cyclohexylpropyl) -His (mono-cyclohexylpropyl) -NH-resin (Rink Amide)
H-His(mono-cyclohexylpropyl)-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-His(mono-cyclohexylpropyl)-OH (분자량 = 501.62 g/mol) (150.49 g, 300 mmol, 3.0 당량) 과 Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 5시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하고, DMF 1000 ml으로 총 6회 레진을 세척한 후 H-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-His(mono-cyclohexylpropyl)-OH (molecular weight = 501.62 g/mol) (150.49 g, 300 mmol, 3.0 equivalent) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution, and the mixture was slowly stirred at room temperature for 5 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of a 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, the resin was treated once more in the same manner, and the resin was washed 6 times with 1000 ml of DMF in total, followed by H-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was obtained (yield >99%).
실시 예 3 : H-Arg(pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl )-NH-레진 (Rink Amide) 제조Example 3: Preparation of H-Arg(pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-Resin (Rink Amide)
H-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-Arg(Pbf)-OH (분자량 = 648.8 g/mol, 194.64 g, 300 mmole)과 Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 5시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반한다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리한다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-Arg(Pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-Arg(Pbf)-OH (molecular weight = 648.8 g/mol, 194.64 g , 300 mmole) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF and added thereto. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution, and the mixture was slowly stirred at room temperature for 5 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of a 20% piperidine/DMF solution is added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Arg(Pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was obtained (yield >99%).
실시 예 4 : Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ 제조Example 4: Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ Manufacturing
H-Arg(Pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-레진(Rink Amide)을 반응기에 투입하고 냉각된 TFA/TIPS/water (95/2.5/2.5) 용액 3 L를 서서히 부어 넣은 다음 상온에서 3시간 동안 교반하였다. 냉각된 디에틸에테르 12 L에 반응액을 서서히 적가하여 펩타이드를 석출시켰다. 실온에서 30분 동안 교반한 다음 여과하여 펩타이드를 회수한 후, 용매를 감압 제거하였다. 진공 건조기에서 5시간 동안 건조 시켜, 백색의 Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ 80.03 g을 수득하였다. (분자량 696.15 g/mol, Crude peptide 순도 65.7%, 수율 115.0%)H-Arg(Pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was put into the reactor and cooled TFA/TIPS/water (95/2.5/2.5) solution 3 L was slowly poured into the mixture and stirred at room temperature for 3 hours. The reaction solution was slowly added dropwise to 12 L of cooled diethyl ether to precipitate the peptide. After stirring at room temperature for 30 minutes and then filtering to recover the peptide, the solvent was removed under reduced pressure. Dry in a vacuum dryer for 5 hours to obtain white Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ 80.03 g was obtained. (Molecular weight 696.15 g/mol, crude peptide purity 65.7%, yield 115.0%)
실시 예 5 : H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ 제조Example 5: Preparation of H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂
[화학식 I][Formula I]
실시예 4에서 수득한 Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ 80.03 g을 정제수에 용해한 다음 0.46μm membrane으로 여과하였다. 여과액을 산업용 HPLC (230 nm, 500 ml/분, 10 미크론 C18 컬럼에서 20분 내에 0.1% TFA 내 아세토니트릴 초기농도 30%에서 65%로 증가)로 반복 주입한 후 main fraction 부분을 분획하여 화학식 I로 표시되는 트라이펩타이드 29.45 g (수율: 36.8%, 순도: 99.21%)을 수득하였다. Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ obtained in Example 4 80.03 g was dissolved in purified water and filtered through a 0.46 μm membrane. After repeated injection of the filtrate by industrial HPLC (230 nm, 500 ml/min, 10 micron C18 column, initial concentration of acetonitrile in 0.1% TFA increased from 30% to 65% within 20 minutes), the main fraction was fractionated and chemical formula 29.45 g of the tripeptide represented by I (yield: 36.8%, purity: 99.21%) was obtained.
수득된 히스티딘 유도체를 포함하는 트라이펩타이드의 분자량 및 순도를 확인하고 그 결과를 도 2 및 도 3에 나타내었다.The molecular weight and purity of the obtained tripeptide containing histidine derivatives were confirmed, and the results are shown in FIGS. 2 and 3 .
실험예 1 : Disk Diffusion AssayExperimental Example 1: Disk Diffusion Assay
상기 실시 예 5에서 제조한 항균 펩타이드의 비브리오균에 대한 항균력을 확인하기 위해 disk diffusion assay를 실시하였다.A disk diffusion assay was performed to confirm the antibacterial activity of the antibacterial peptide prepared in Example 5 against Vibrio.
보다 구체적으로 실시예 5에서 제조된 화학식 I로 표시되는 트라이펩타이드 (WP-Vib1로 표시)(2 mM), 대조군으로 DMSO, H2O 및 gentamicin (2 mM) 20μL을 각각 6mm paper disc에 흡수시킨 후 건조시켰다. 다음으로 1.5×108cfu의 비브리오 파라해모리티쿠스 (Vibrio parahaemolyticus)(생물자원센터, KCTC 2471)을 LB Agar 배지에 도말한 후, 건조된 paper disc를 배지에 올려놓고, 37.5℃에서 18시간 동안 배양하였다. More specifically, the tripeptide represented by Formula I prepared in Example 5 (represented as WP-Vib1) (2 mM), DMSO, H 2 O, and gentamicin (2 mM) as a control, 20 μL, respectively, were absorbed into a 6 mm paper disc. then dried. Next, 1.5 × 10 8 cfu of Vibrio parahaemolyticus (Biological Resources Center, KCTC 2471) was spread on the LB Agar medium, and then the dried paper disc was placed on the medium and maintained at 37.5 ° C for 18 hours. cultured for a while.
그 결과, 도 4에 나타난 바와 같이, 실시예 5에서 제조된 화학식 I로 표시되는 트라이펩타이드 (WP-Vib1로 표시)는 양성 대조군인 gentamicin과 유사하게 비브리오 파라해모리티쿠스 (Vibrio parahaemolyticus)에 대한 항균활성을 가지고 있어 균 성장을 저해한다는 것을 알 수 있었다.As a result, as shown in Figure 4, the tripeptide represented by Formula I prepared in Example 5 (represented by WP-Vib1) is similar to gentamicin, which is a positive control, for Vibrio parahaemolyticus . It was found that it had antibacterial activity and inhibited the growth of bacteria.
실험예 2 : Alamar blue assayExperimental Example 2: Alamar blue assay
상기 실시 예 5에서 제조한 항균 펩타이드의 비브리오균에 대한 항균력을 확인하기 위해 alamar blue assay를 실시하였다.In order to confirm the antibacterial activity of the antibacterial peptide prepared in Example 5 against Vibrio, alamar blue assay was performed.
106개의 비브리오 파라해모리티쿠스 (Vibrio parahaemolyticus)(생물자원센터, KCTC 2471) 배양액 80μL 및 Alamar Blue(10x) 10μL를 96 well plate의 각 well에 넣고, shaking incubator에서 1시간 배양하였다. 배양 후, 각 well에 시료를 10μL씩 처리하고, Fluorescence plate reader (Victor3, PerkinElmer)를 이용하여 530 nm에서 excitation 590 nm에서의 emission 조건으로 15분 간격으로 형광을 측정하였다. 10 6 Vibrio parahaemolyticus (Center for Biological Resources, KCTC 2471) 80 μL of culture medium and 10 μL of Alamar Blue (10x) were put into each well of a 96 well plate, and cultured in a shaking incubator for 1 hour. After incubation, 10 μL of the sample was treated in each well, and fluorescence was measured at 15-minute intervals under emission conditions at 530 nm and excitation at 590 nm using a fluorescence plate reader (Victor3, PerkinElmer).
이때, 시료는 gentamicin (농도:0.016~2mM), 트라이펩타이드 (농도:0.016~2mM)(WP-Vib1로 표시), 실시예 5에서 제조된 화학식 I로 표시되는 트라이펩타이드(농도:0.016~2mM)(WP-Vib1로 표시)를 이용하였다.At this time, the sample is gentamicin (concentration: 0.016 ~ 2mM), tripeptide (concentration: 0.016 ~ 2mM) (represented by WP-Vib1), tripeptide represented by Formula I prepared in Example 5 (concentration: 0.016 ~ 2mM) (denoted as WP-Vib1) was used.
Effective concentraion (EC50)값은 Graphpad prism 소프트웨어 (version 4.0)를 이용하여 계산하고, 그 결과를 도 5에 나타내었다. Effective concentration (EC 50 ) values were calculated using Graphpad prism software (version 4.0), and the results are shown in FIG. 5 .
도 5에 나타난 바와 같이, 실시예 5에서 제조된 화학식 I로 표시되는 트라이펩타이드 (농도:0.016~2mM)(WP-Vib1로 표시)는 양성 대조군인 gentamicin과 유사하게 비브리오 파라해모리티쿠스 (Vibrio parahaemolyticus)에 대한 항균활성을 갖는 것을 알 수 있었다.As shown in Figure 5, the tripeptide represented by Formula I prepared in Example 5 (concentration: 0.016 ~ 2 mM) (represented as WP-Vib1) is similar to the positive control gentamicin, Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) was found to have antibacterial activity.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당 업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As above, specific parts of the present invention have been described in detail, and it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (6)
[화학식 I]
A tripeptide comprising a histidine derivative having the structure of Formula I below.
[Formula I]
An antibacterial composition containing a tripeptide containing a histidine derivative having the structure of claim 1 as an active ingredient.
The antimicrobial composition according to claim 2, wherein the antimicrobial composition has antibacterial activity against Vibrio bacteria.
(b) 상기 단계 (a)에서 수득한 레진에 Fmoc-His(mono-cyclohexylpropyl)-OH 및 Fmoc-Arg(보호기)-OH를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득하는 단계;
(c) 상기 단계 (b)에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 트라이펩타이드를 수득하는 단계; 및
(d) 상기 단계 (c)에서 수득한 비정제 트라이펩타이드를 정제하여 화학식 I로 표시되는 트라이펩타이드를 수득하는 단계로 구성된 히스티딘 유도체를 포함하는 트라이펩타이드의 제조방법.
[화학식 1]
[화학식 2]
[화학식 3]
[화학식 I]
(a) obtaining Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by Formula 1 by a solid-phase synthesis method;
(b) Resin obtained by sequentially combining Fmoc-His (mono-cyclohexylpropyl) -OH and Fmoc-Arg (protecting group) -OH to the resin obtained in step (a) to which the protected peptide represented by Formula 2 is bound Obtaining;
(c) obtaining a non-purified tripeptide represented by Chemical Formula 3 by simultaneously removing the protecting group of the resin and amino acid from the resin to which the protected peptide obtained in step (b) is bound; and
(d) Purifying the non-purified tripeptide obtained in step (c) to obtain a tripeptide represented by formula (I).
[Formula 1]
[Formula 2]
[Formula 3]
[Formula I]
The method of claim 4, wherein the resins are Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin. Resin) method for producing a tripeptide, characterized in that selected from the group consisting of.
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