KR100991096B1 - A Method for preparing octreotide using by solid phase synthesis - Google Patents
A Method for preparing octreotide using by solid phase synthesis Download PDFInfo
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- KR100991096B1 KR100991096B1 KR1020100002115A KR20100002115A KR100991096B1 KR 100991096 B1 KR100991096 B1 KR 100991096B1 KR 1020100002115 A KR1020100002115 A KR 1020100002115A KR 20100002115 A KR20100002115 A KR 20100002115A KR 100991096 B1 KR100991096 B1 KR 100991096B1
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- KR
- South Korea
- Prior art keywords
- fmoc
- fluorenylmethoxycarbonyl
- resin
- reaction
- group
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- 238000000034 method Methods 0.000 title claims abstract description 38
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- 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
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
본 발명은 약리활성을 갖는 펩타이드인 옥트레오타이드(octreotide)의 제조방법에 관한 것으로, 본 발명의 제조방법은 고분자 지지체 상에서 아미노산을 순차적으로 결합시킨 후 최종적으로 고분자 지지체에서 유리시켜 고순도로 펩타이드를 제조할 수 있는 새로운 펩타이드 제조방법에 관한 것이다.The present invention relates to a method for preparing octreotide, which is a peptide having pharmacological activity, and the method of the present invention binds amino acids sequentially on a polymer support, and finally releases it from the polymer support to produce a peptide with high purity. It relates to a new peptide production method that can be.
Description
본 발명은 약리 활성을 갖는 펩타이드인 옥트레오타이드(octreotide)의 제조방법에 관한 것이다.The present invention relates to a method for preparing octreotide, which is a peptide having pharmacological activity.
[문헌 1] Lloyd, et al., Am . J. Physiol . 268, p.G102, 1995Lloyd, et al., Am . J. Physiol . 268 , p.G102, 1995
[문헌 2] Clark, R.V. et al ., Clin . Res . 38, p.9943A, 1990[Reference 2] Clark, RV et al ., Clin . Res . 38 , p.9943A, 1990
[문헌 3] Ambrosi, B., et al ., Acta Endocr .( Copenh .) 122, p.569-576, 1990[3] Ambrosi, B., et al ., Acta Endocr . ( Copenh .) 122 , p . 569-576, 1990
[문헌 4] Miller, D., et al ., Canad . Med . Ass . J. 145, p.227-228, 1991Document 4 Miller, D., et al ., Canad . Med . Ass . J. 145 , p. 227-228, 1991
[문헌 5] Palmieri, G,M.A., et al ., J. Bone & Mineral Res . 7( Suppl .1), p.S240(Abs. 591), 1992Document 5 Palmieri, G, MA, et al ., J. Bone & Mineral Res . 7 ( Suppl. 1) , p. S240 (Abs. 591), 1992
[문헌 6] Koberstein, B. et al ., Gastroenterology 28, p.295-301, 1990Document 6 Koberstein, B. et al ., Gastroenterology 28 , p . 295-301, 1990
[문헌 7] Laron, Z., Israel J. Med . Sci . 26(1), p.1-2, 19907 Laron, Z., Israel J. Med . Sci . 26 (1) , p. 1-2 , 1990
[문헌 8] Bauer, F.E., et al ., Europ . J. Pharmacol . 183, p.55, 1990[Reference 8] Bauer, FE, et al ., Europ . J. Pharmacol . 183 , p. 55, 1990
[문헌 9] Mozell, E., et al ., Surg . Gynec . Obstet . 170, p.476-484, 19909 Mozell, E., et al ., Surg . Gynec . Obstet . 170 , p.476-484, 1990
[문헌 10] AIDS, Cello, J. P., et al ., Gastroenterology 98(5), Part 2, Suppl., p.A163, 1990[Reference 10] AIDS, Cello, JP, et al ., Gastroenterology 98 (5) , Part 2, Suppl., p. A163, 1990
[문헌 11] Alhindawi, R., et al ., Can . J. Surg . 33, p.139-142, 1990[11] Alhindawi, R., et al ., Can . J. Surg . 33 , p. 139-142, 1990
[문헌 12] Petrelli, N., et al ., Proc . Amer . Soc . Clin . Oncol . 10, p.138(Abs.417) 199112. Petrelli, N., et al ., Proc . Amer . Soc . Clin . Oncol . 10 , p. 138 (Abs. 417) 1991
[문헌 13] Tulassay, Z., et al ., Gastroenterology 98(5), Part 2, Suppl., p.A238, 199013 Tulassay, Z., et al ., Gastroenterology 98 (5) , Part 2, Suppl., p. A238, 1990
[문헌 14] Fedorak, R. N., et al ., Can . J. Gastroenterology 3(2), p.53- 57, 1989Document 14 Fedorak, RN, et al ., Can . J. Gastroenterology 3 (2) , p. 53- 57, 1989
[문헌 15] Soudah, H., et al ., Gastroenterology 98(5), Part 2, Suppl., p.A129, 199015 Soudah, H., et al ., Gastroenterology 98 (5) , Part 2, Suppl., p. A129, 1990
[문헌 16] Modigliani, E., et al ., Ann . Endocr . ( Paris ) 50, p.483-488, 198916. Modigliani, E., et al ., Ann . Endocr . ( Paris ) 50 , p.483-488, 1989
[문헌 17] Camisa, C., et al ., Cleveland Clinic J. Med . 57(1), p.71-76, 1990Reference 17 Camisa, C., et al ., Cleveland Clinic J. Med . 57 (1) , p. 71-76, 1990
[문헌 18] Kooner, J. S., et al ., Brit . J. Clin . Pharmacol ., 28, p.735-736, 1989) Document 18 Kooner, JS, et al ., Brit . J. Clin . Pharmacol ., 28, p . 735-736, 1989)
[문헌 19] Abelson, J. L., et al ., Clin . Psychopharmacol . 10, p.128-132, 1990[19] Abelson, JL, et al ., Clin . Psychopharmacol . 10 , p. 128-132, 1990
[문헌 20] Soudah, H., et al ., Clin . Res . 39, p.303A, 1991Soudah, H., et al ., Clin . Res . 39 , p.303A, 1991
[문헌 21] Nott, D. M., et al ., Brit . J. Surg . 77, p.A691, 1990[21] Nott, DM, et al ., Brit . J. Surg . 77 , p. A691, 1990
[문헌 22] Branch, M. S., et al ., Gastroenterology 100(5), Part 2 Suppl., p.A425, 199122, Branch, MS, et al ., Gastroenterology 100 (5) , Part 2 Suppl., p. A425, 1991
[문헌 23] Hasler, W., et al ., Gastroenterology 100(5), Part 2, Suppl., p.A448, 199123. Hasler, W., et al ., Gastroenterology 100 (5) , Part 2, Suppl., p. A448, 1991
[문헌 24] Chang, T. C., et al ., Brit . Med . J. 304, p.158, 1992[Reference 24] Chang, TC, et al ., Brit . Med . J. 304 , p. 158, 1992
[문헌 25] Prelevic, G. M., et al ., Metabolism Clinical and Experimental 41, Suppl. 2, p.76-79, 1992[25] Prelevic, GM, et al ., Metabolism Clinical and Experimental 41 , Suppl. 2, p. 76-79, 1992
[문헌 26] Jenkins, S. A., et al ., Gut . 33, p.404-407, 1992[26] Jenkins, SA, et al ., Gut . 33 , p. 404-407, 1992
[문헌 27] Koper, J. W., et al ., J. Clin . Endocr . Metab . 74, p.543-547, 1992Document 27 Koper, JW, et al ., J. Clin . Endocr . Metab . 74 , p.543-547, 1992
[문헌 28] Bartlett, D. L., et al ., Surg . Forum . 42, p.14-16, 1991[Reference 28] Bartlett, DL, et al ., Surg . Forum . 42 , p. 14-16, 1991
[문헌 29] Ezzat S., et al ., Ann Intern Med . 117, p.711-718, 1992 29 Ezzat S., et al ., Ann Intern Med . 117 , p.711-718, 1992
[문헌 30] Flogstad A K, et al ., J Clin Endocrinol Metab . 82, p.23-28, 1997Document 30 Flogstad AK, et al ., J Clin Endocrinol Metab . 82 , p. 23-28, 1997
[문헌 31] Bodanszky et al ., Synthetic Peptides : A User's Guide, G.R. Grant, ed., Freeman & Co., p.77-183, 1992[31] Bodanszky et al ., Synthetic Peptides : A User's Guide, GR Grant, ed., Freeman & Co., p. 77-183, 1992
[문헌 32] 미국등록특허 제 6,664,372호32. US Patent No. 6,664,372
[문헌 33] 미국등록특허 제 6,624,290호[Reference 33] US Patent No. 6,624,290
[문헌 34] Tamamura, H. et al. Int . J. Pept . Pro . Res. 45, 321, 1995 34 Tamamura, H. et al . Int . J. Pept . Pro . Res . 45 , 321, 1995
[문헌 35] Albericio, F. et al . In "Fmoc Solid Phase Synthesis: A Practical Approach", Chan, W.C. & White, P.D.(Eds.), Oxford Univ. Press, Oxford, 2000, 104Document 35 Albericio, F. et al . In "Fmoc Solid Phase Synthesis: A Practical Approach", Chan, WC & White, PD (Eds.), Oxford Univ. Press, Oxford, 2000, 104
[문헌 36] Beekman, N.J.C.M. et al. J. Pept . Res. 50, 357, 1997[36] Beekman, NJCM et al . J. Pept . Res . 50 , 357, 1997
[문헌 37] Albericio, F. et al. Int . J. Pept . Pro . Res. 37, 402, 199137 Albericio, F. et al . Int . J. Pept . Pro . Res . 37 , 402, 1991
[문헌 38] Ranganathan, S & Jayaraman, N. J. Chem . Soc ., Chem . Commun . 934, 199138 Ranganathan, S & Jayaraman, N. J. Chem . Soc , Chem . Commun . 934, 1991
본 발명은 약리활성을 갖는 펩타이드인 옥트레오타이드(octreotide)의 제조방법에 관한 것이다.The present invention relates to a method for preparing octreotide, which is a peptide having pharmacological activity.
소마토스타틴은 브라제아우 등에 의해 발견된, 14개의 아미노산으로 구성된 펩타이드로서 뇌하수체, 췌장, 위장관 등과 같은 조직 내에서의 분비 과정에 강력한 억제 작용이 있음이 알려져 있다. 또한 소마토스타틴은 중추신경계에서 신경조절체로도 작용한다. 억제 작용에 있어 소마토스타틴의 생물학적 효과는 G 단백질 결합 수용체에 기인하는데, 여기에는 5종의 아류형(subtype)이 존재한다.Somatostatin is a peptide consisting of 14 amino acids found by Brazeau et al. It is known that there is a strong inhibitory effect on the secretion process in tissues such as the pituitary gland, pancreas, gastrointestinal tract. Somatostatin also acts as a neuromodulator in the central nervous system. The biological effect of somatostatin in the inhibitory action is attributable to the G protein binding receptor, which has five subtypes.
소마토스타틴은 호르몬 분비, 글루카곤, 인슐린, 아밀린, 신경전달물질 분비에 관여한다. 그 중 몇 가지는 소마토스타틴의 특이적 수용체와 연관되어 있는데, 예를 들면 성장 호르몬의 억제는 소마토스타틴 타입-2 수용체, 인슐린 억제는 타입-5 수용체와 연관되어있다(Lloyd, et al., Am. J. Physiol. 268, G102, 1995).Somatostatin is involved in hormone secretion, glucagon, insulin, amylin, and neurotransmitter secretion. Some of them are associated with specific receptors for somatostatin, for example growth hormone suppression is associated with somatostatin type-2 receptors and insulin inhibition is associated with type-5 receptors (Lloyd, et al., Am. J. Physiol. 268, G102, 1995).
소마토스타틴은 다양한 질병의 처치에 사용되는데, 하기의 문헌들을 통해 확인할 수 있다: 쿠싱 신드롬(Clark, R.V. et al., Clin. Res. 38, 9943A, 1990); 생식샘수암(Ambrosi, B., et al., Acta Endocr. (Copenh.) 122, 569-576, 1990); 고부갑상선(Miller, D., et al., Canad. Med. Ass. J., 145, 227-228, 1991); 파제의 질병(Palmieri, G,M.A., et al., J. Bone & Mineral Res, 7 (Suppl.1), S240(Abs. 591), 1992); 바이포마(Koberstein, B. et al., Gastroenterology, 28, 295-301, 1990); 고인슐린증(Laron, Z., Israel J. Med. Sci., 26(1), 1-2, 1990); 가스트리노마 (Bauer, F.E., et al., Europ. J. Pharmacol., 183, 55, 1990); 조린거-엘리슨 신드롬(Mozell, E., et al ., Surg. Gynec. Obstet., 170, p.476-484, 1990); 에이즈 및 기타 상태에 따른 과민성 설사증(AIDS, Cello, J. P., et al ., Gastroenterology, 98(5), Part 2, Suppl., A163 1990); 증가된 가스트린-유리 펩타이드(Alhindawi, R., et al ., Can. J. Surg., 33, 139-142, 1990); 화학치료와 동반된 설사증(Petrelli, N., et al ., Proc. Amer. Soc. Clin. Oncol., 10, 138(Abs.417) 1991); 췌장염(Tulassay, Z., et al ., Gastroenterology, 98(5), Part 2, Suppl., A238, 1990); 크론의 질병(Fedorak, R. N., et al ., Can. J. Gastroenterology, 3(2), 53- 57, 1989); 전신 경화(Soudah, H., et al ., Gastroenterology, 98(5), Part 2, Suppl., A129, 1990); 갑상선암(Modigliani, E., et al ., Ann., Endocr. (Paris), 50, 483-488, 1989); 건선(Camisa, C., et al., Cleveland Clinic J. Med., 57(1), 71-76, 1990); 저혈압(Kooner, J. S., et al., Brit. J. Clin. Pharmacol., 28, 735-736, 1989); 패닉(Abelson, J. L., et al ., Clin. Psychopharmacol., 10, 128-132, 1990); 피부경화증(Soudah, H., et al ., Clin. Res., 39, 303A, 1991); 작은창자 장애(Nott, D. M., et al ., Brit. J. Surg., 77, A691, 1990); 위식도 역류(Branch, M. S., et al., Gastroenterology, 100(5), Part 2 Suppl., p. A425, 1991); 십이지장 역류(Hasler, W., et al., Gastroenterology, 100(5), Part 2, Suppl., p.A448, 1991); 그레이브의 질병(Chang, T. C., et al ., Brit. Med. J., 304, 158, 1992); 다낭성 난소질병(Prelevic, G. M., et al ., Metabolism Clinical and Experimental, 41, Suppl. 2, 76-79, 1992); 위장출혈(Jenkins, S. A., et al ., Gut., 33, p.404-407, 1992); 백혈병 및 수막종(Koper, J. W., et al ., J. Clin. Endocr. Metab., 74, p.543-547, 1992); 암 카헥시(Bartlett, D. L., et al ., Surg. Forum., 42, p.14-16, 1991).Somatostatin is used to treat a variety of diseases, which can be found in the following literature: Cushing syndrome (Clark, R.V.et al., Clin. Res. 38, 9943A, 1990); Gonadal carcinoma (Ambrosi, B.,et al., Acta Endocr. (Copenh. 122, 569-576, 1990); Hyperthyroidism (Miller, D.,et al., Canad. Med. Ass. J., 145, 227-228, 1991); Faget's disease (Palmieri, G, M.A.,et al., J. Bone & Mineral Res, 7 (Suppl. 1), S240 (Abs. 591), 1992); Koberstein, B.et al., Gastroenterology, 28, 295-301, 1990); Hyperinsulinemia (Laron, Z., Israel J. Med. Sci., 26 (1), 1-2, 1990); Gastrinoma (Bauer, F.E.,et al., Europ. J. Pharmacol., 183, 55, 1990); Joringer-Elison syndrome (Mozell, E.,et al ., Surg. Gynec. Obstet., 170, p. 476-484, 1990); Irritable diarrhea due to AIDS and other conditions (AIDS, Cello, J. P.,et al ., Gastroenterology, 98 (5), Part 2, Suppl., A163 1990); Increased gastrin-free peptide (Alhindawi, R.,et al ., Can. J. Surg., 33, 139-142, 1990); Diarrhea with chemotherapy (Petrelli, N.,et al ., Proc. Amer. Soc. Clin. Oncol., 10, 138 (Abs. 417) 1991); Pancreatitis (Tulassay, Z.,et al ., Gastroenterology, 98 (5), Part 2, Suppl., A238, 1990); Crohn's disease (Fedorak, R. N.,et al ., Can. J. Gastroenterology, 3 (2), 53-57, 1989); Systemic hardening (Soudah, H.,et al ., Gastroenterology, 98 (5), Part 2, Suppl., A129, 1990); Thyroid cancer (Modigliani, E.,et al ., Ann., Endocr. (Paris), 50, 483-488, 1989); Psoriasis (Camisa, C., et al., Cleveland Clinic J. Med., 57 (1), 71-76, 1990); Hypotension (Kooner, J. S.,et al., Brit. J. Clin. Pharmacol., 28, 735-736, 1989); Panic (Abelson, J. L.,et al ., Clin. Psychopharmacol., 10, 128-132, 1990); Scleroderma (Soudah, H.,et al ., Clin. Res., 39, 303A, 1991); Small bowel disorders (Nott, D. M.,et al ., Brit. J. Surg., 77, A691, 1990); Gastroesophageal reflux (Branch, M. S., et al., Gastroenterology, 100 (5), Part 2 Suppl., P. A425, 1991); Duodenal reflux (Hasler, W., et al., Gastroenterology, 100 (5), Part 2, Suppl., P. A448, 1991); Grave's disease (Chang, T. C.,et al ., Brit. Med. J., 304, 158, 1992); Polycystic ovarian disease (Prelevic, G. M.,et al ., Metabolism Clinical and Experimental, 41, Suppl. 2, 76-79, 1992); Gastrointestinal bleeding (Jenkins, S. A.,et al ., Gut., 33, p. 404-407, 1992); Leukemia and meningioma (Koper, J. W.,et al ., J. Clin. Endocr. Metab., 74, p. 543-547, 1992); Am carhex (Bartlett, D. L.,et al ., Surg. Forum., 42, p. 14-16, 1991).
말단비대증 환자에 대한 소마토스타틴 계 약물인 옥트레오타이드(octreotide)와 란레오타이드(lanreotide)의 효능은 이미 잘 알려져 있으며(Ezzat S., et al ., Ann Intern Med. 117, 711-718 , 1992), 이들의 서방형 제제를 사용하여 개선된 결과도 보고된바 있다(Flogstad A K, et al ., J Clin Endocrinol Metab. 82, 23-28, 1997).The efficacy of octreotide and lanreotide, the somatostatin-based drugs in acromegaly patients, is well known (Ezzat S., et. al ., Ann Intern Med. 117, 711-718, 1992), and improved results using their sustained release formulations have also been reported (Flogstad AK, et. al ., J Clin Endocrinol Metab. 82, 23-28, 1997).
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, John Wiley & Sons, 1976).Chemical synthesis of peptides can be largely divided into solution phase synthesis and solid phase synthesis. Solution-phase synthesis is a classical chemical synthesis method in which all reagents are dissolved in a solution. The reaction rate is fast, but separation and purification are difficult. Solid phase synthesis has been developed since RB Merrifield's theory of solid phase peptide synthesis has been developed and has the advantage of easy separation and automation (Bodanszky et. al ., In Peptide Synthesis, John Wiley & Sons, 1976).
메리필드의 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-클로로 트리틸 수지와 펩타이드의 카르복실 말단을 아미드 형태로 얻을 수 있는 링크아미드 수지가 대표적 수지이다.Since Merrifield's research, numerous peptide synthesis resins have been developed and many peptides have been synthesized using them. Chloromethyl polystyrene resins developed by Merrifield and Wang resins with the structure of 4-alkoxybenzyl alcohol, which were developed to compensate for the shortcomings of this resin, were developed relatively early. It became. Since these resins have been continuously developed to compensate for the shortcomings, the typical resin is a 2-chloro trityl resin with a trityl structure and a linkamide resin that can obtain the carboxyl terminal of the peptide in the amide form.
이들 수지를 이용하여 초기에는 간단한 펩타이드들이 합성되었으나 점차 생리활성을 갖는 복잡한 펩타이드들이 합성되었다. 비천연 아미노산을 포함하는 펩타이드들은 효소에 의한 천연 합성이 불가능하므로 화학적 합성에 의해 제조되었고, 이 중 D-아미노산이나 아자아미노산(aza-amino acid)를 포함하는 펩타이드 중에는 강력한 생리활성 작용으로 의약품으로 사용되는 것들이 있어 많은 연구가 진행되었다(미국등록특허 제6,664,372호; 미국등록특허 제6,624,290호). Initially, simple peptides were synthesized using these resins, but complex peptides having physiological activity were synthesized. Peptides containing non-natural amino acids were manufactured by chemical synthesis because they cannot be synthesized naturally by enzymes, and among them, peptides containing D-amino acid or azaamino acid are used as medicines due to their strong physiological activity. Many studies have been carried out because of such things (US Patent No. 6,664,372; US Patent No. 6,624,290).
이에 본 발명자들은 아민 말단 및 측쇄가 모두 보호된 아미노산 유도체들을 고분자 지지체와 순차적으로 결합시켜 펩타이드를 제조하는 방법으로 특히, 시스테인(Cys)의 측쇄를 고분자 지지체에 고정하는 방법을 사용하여 펩타이드를 고순도 및 고수율로 제조할 수 있음을 발견하여 본 발명을 완성하였다.Accordingly, the present inventors have prepared a peptide by sequentially binding amino acid derivatives protected both at the amine end and the side chain with a polymer support. In particular, the inventors fixed the peptide using the method of fixing the side chain of cysteine (Cys) to the polymer support. The present invention has been completed by discovering that it can be produced in high yield.
상기 목적을 달성하기 위하여, 본 발명은 고분자 지지체 수지에 시스테인의 측쇄 또는 C-말단을 결합시키는 제 1단계; 상기 수지에서 아미노산 유도체를 순차적으로 결합시키는 펩타이드를 합성하는 제 2단계; 상기 합성 펩타이드가 결합된 수지로부터 펩타이드의 유리 및 디설피드 사슬을 통한 고리화 반응을 반응 용매하에서 수행하는 제 3단계의 공정을 포함함을 특징으로 하는 하기 구조식 (a)의 소마토스타틴 또는 구조식 (b)의 옥트레오타이드를 제조하는 제조 방법을 제공한다.
In order to achieve the above object, the present invention comprises a first step of bonding the side chain or C-terminus of cysteine to the polymer support resin; A second step of synthesizing a peptide that sequentially binds amino acid derivatives in the resin; Somatostatin or structural formula (b) of the following structural formula (a), comprising the step of performing a cyclization reaction via free and disulfide chains of the peptide from the resin to which the synthetic peptide is bound in a reaction solvent It provides a manufacturing method for producing an octreotide.
(a) 소마토스타틴
(a) somatostatin
(b) 옥트레오타이드
(b) octreotide
본원에서 정의되는 서열에서, Ala는 알라닌(alanine), Gly는 글리(glycine), Cys는 시스테인(cysteine), Lys는 리신(lysine), Asn는 아스파라긴(asparganine), Phe는 페닐알라닌(phenylalanine), Trp는 트립토판(tryptophan), Thr는 트레오닌(threonine), Ser는 세린(serine), DPhe는 D-형 광학활성을 갖는 페닐알라닌, DTrp는 D-형 광학활성을 갖는 트립토판, Thr-ol은 트레오니놀로 트레오닌의 C-말단이 알코올 형태로 환원된 것을 의미한다.In the sequences defined herein, Ala is alanine, Gly is glycine, Cys is cysteine, Lys is lysine, Asn is asparganine, Phe is phenylalanine, Trp Is tryptophan, Thr is threonine, Ser is serine, DPhe is phenylalanine with D-type optical activity, DTrp is tryptophan with D-type optical activity, Thr-ol is threoninolo threonine Means that the C-terminus of is reduced to the alcohol form.
본원에서 정의되는 고분자 지지체 수지는 폴리스티렌, 폴리아미드, 유리 또는 실리카로 구성된 군으로부터 선택되며, 바람직하게는 폴리스티렌인 것을 특징으로 한다.The polymeric support resin as defined herein is selected from the group consisting of polystyrene, polyamide, glass or silica, and is preferably characterized in that it is polystyrene.
본원에서 정의되는 아미노산 유도체는 아민 말단 및 측쇄가 Boc(tert-butoxycarbonyl), Fmoc(9-fluorenylmethoxycarbonyl), Cbz(benzyloxycarbonyl), tBu(tert-butyl), StBu(tert-butylthio), Trt(tri-phenylmethyl; trityl), Acm(acetamidomethyl) 또는 Tacm(trimethylacetamido-methyl)의 보호기로부터 선택되며, 바람직하게는 Fmoc(9-fluorenylmethoxycarbonyl) 보호기로 보호된 것임을 특징으로 한다.The amino acid derivatives as defined herein have amine ends and side chains of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), tBu (tert-butyl), StBu (tert-butylthio), and Trt (tri-phenylmethyl). trityl), Acm (acetamidomethyl) or Tacm (trimethylacetamido-methyl) protecting group, and is preferably characterized by being protected with a Fmoc (9-fluorenylmethoxycarbonyl) protecting group.
상기 디설피드 사슬을 통한 고리화 반응은 TFA(trifluoroacetic acid), TFMSA(trifluoromethanesulfonic acid), Hg(수은) 2가 이온, Ag(은) 1가 이온, 요오드, Tl(탈륨) 3가 이온 또는 티올에서 선택된 시약으로 반응시키는 것을 특징으로 하는 제조방법을 제공한다.The cyclization reaction through the disulfide chain is carried out in trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TFMSA), Hg (mercury) divalent ion, Ag (silver) monovalent ion, iodine, Tl (thallium) trivalent ion or thiol. It provides a production method characterized in that the reaction with the selected reagent.
본 발명의 반응용매로는 화학반응에서 일반적으로 사용되는 디클로로메탄, 클로로포름, 디클로로에탄, 디메틸포름아미드, 디메틸아세트아미드, N-메틸피롤리디논, 테트라히드로푸란, 디옥산 또는 이들의 혼합용매를 반응용매로 사용하며, 바람직하게는, 디클로로메탄, 디메틸포름아미드, 트리플루오르아세트산이다. As the reaction solvent of the present invention, dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, N-methylpyrrolidinone, tetrahydrofuran, dioxane, or a mixed solvent thereof generally used in chemical reactions is reacted. It is used as a solvent, Preferably it is dichloromethane, dimethylformamide, trifluoroacetic acid.
본원에 개시된 반응들의 반응 온도는 그 제한은 없으나, 바람직하게는 0℃ 내지 70℃, 보다 바람직하게는 20℃ 내지 50℃의 범위이다. 반응시간은 10분 내지 48시간의 범위이며, 바람직하게는 각 반응물질의 반응성과 반응 후 생성물의 생산성을 고려할 때, 1시간 내지 24시간의 범위이다. 단, 원하는 만큼 반응이 진행되지 않았을 경우에는 동일 반응을 2회 내지 5회 더 수행하여 반응 수율을 높일 수 있다.The reaction temperature of the reactions disclosed herein is not limited, but is preferably in the range of 0 ° C to 70 ° C, more preferably 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, considering the reactivity of each reactant and the productivity of the product after the reaction. However, when the reaction does not proceed as desired, the same reaction may be performed 2 to 5 times more to increase the reaction yield.
또한 본원에 개시된 상기 공정은 상기 제 2단계 이후 필요에 따라 용액상에서 아미노산을 결합할 수 있음을 특징으로 한다.The process disclosed herein is also characterized in that it is possible to bind amino acids in solution as necessary after the second step.
본 발명의 목적은 약리 활성을 갖는 펩타이드인 소마토스타틴 또는 그 유사체인 옥트레오타이드의 제조방법을 제공하는 것으로, 하기의 반응식들에 도시된 방법에 의해 화학적으로 합성될 수 있지만, 이들 예로만 한정되는 것은 아니다. SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing somatostatin, which is a peptide having pharmacological activity, or an analog thereof, octretide, which may be chemically synthesized by the method shown in the following schemes, but is not limited thereto. no.
하기의 반응식들은 본 발명의 화합물의 제조방법을 제조 단계별로 나타내는 것으로 본 발명의 화합물은 반응식의 합성과정에서 사용되는 시약, 용매 및 반응 순서를 바꾸는 등의 작은 변경으로 제조될 수 있다.The following schemes represent the preparation steps of the compounds of the present invention in stages of preparation. The compounds of the present invention may be prepared with minor modifications, such as changing the reagents, solvents, and reaction sequences used in the synthesis of the schemes.
먼저 본 발명의 반응 단계는 하기한 반응식에 기재된 도식에 의하여 설명된다.
First, the reaction step of the present invention is illustrated by the scheme described in the following scheme.
(반응식1)(Scheme 1)
구체적으로, 상기 반응식 1은 소마토스타틴의 합성에 대한 것이다. 본원 발명은 상기 반응식 1에서 보는 바와 같이, 먼저, 아민 말단이 Fmoc (9-fluorenylmethoxycarbonyl) 기로 보호된 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH 등과 같은 시스테인 유도체를 2-클로로트리틸 클로라이드 수지와 DMF(dimethyl formamide) 또는 DCM(dichloromethane) 등의 용매, DIEA(N,N-diisopropylethylamine) 등의 염기하에서 결합시켜 아미노산이 결합된 수지를 제조하는 제 1단계; 상기 단계의 활성형 수지에 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA(dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 제 2단계; 상기 탈보호화된 수지에 아민 말단 및 측쇄가 모두 보호기로 보호된 아미노산 유도체들인 Fmoc(9-fluorenylmethoxycarbonyl) -Ser(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Trp(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Asn(Trt)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Gly-OH, Boc-Ala-OH을 DIC(diisopropyl carbodiimide) 또는 HOBt(N-hydroxylbenzotriazole)와 같은 반응시약으로 순차적으로 첨가한 후, 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA(dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 단계를 반복하는 일반적인 고체상 펩타이드 합성법에 따라, 펩타이드를 합성하여 직쇄(straight chain) 형태의 펩타이드-수지 연결체를 얻는 제 3단계; 상기 연결체에 DMF/아니솔 용액, Tl(tfa)3(thalium trifluoroacetate) 혼합액, 요오드, Hg(수은) 2가 이온 또는 Ag(은) 1가 이온 등의 설피드기 형성시약을 첨가하여 수지 상에서 이황화 브리지(disulfide bridge) 형성을 통한 고리화 반응을 시키는 제 4단계; 상기 연결체에 TFA(trifluoroacetic acid): TIS(triisopropylsilane) 혼합수용액 또는 DCM(dichloromethane) 등의 약산성 분해(cleavage) 용액을 첨가하여 측쇄 보호기를 제거함과 동시에 수지로부터 절단하는 제 5단계를 포함하는 공정을 통하여 본 발명의 소마토스타틴을 제조할 수 있다. 상기의 PG는 보호기(protecting group)를 의미한다.
Specifically, Scheme 1 relates to the synthesis of somatostatin. The present invention, first, an amine-terminal Fmoc (9-fluorenylmethoxycarbonyl) of Fmoc (9-fluorenylmethoxycarbonyl) protected with -Cys (Acm) 2- chloro trityl chloride resin cysteine derivatives such as -OH as shown in Scheme 1 And a first step of preparing an amino acid-bonded resin by binding under a solvent such as DMF (dimethyl formamide) or DCM (dichloromethane) or DIEA (N, N-diisopropylethylamine); Fmoc (9-fluorenylmethoxycarbonyl) protecting group is removed by a deprotection reaction which is treated with a reaction solvent such as piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) in the active resin of the step. A second step of doing; The Talbot the resin is expensive amines, which are both ends and side chains of the amino acid derivative protected by a protective group Fmoc (9-fluorenylmethoxycarbonyl) -Ser ( tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu) -OH, Fmoc (9 -fluorenylmethoxycarbonyl) -Phe -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Trp (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc (9-fluorenylmethoxycarbonyl) -Asn (Trt) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Gly-OH, Boc-Ala-OH in order with reaction reagents such as DIC (diisopropyl carbodiimide) or HOBt (N-hydroxylbenzotriazole) Following the addition, piperidine / DMF (dimethyl formamide) (20:80 , v / v) or a de-protecting reaction to treatment in a reaction solvent such as DMA (dimethylacetamide) Fmoc (9- fluorenylmethoxycarbonyl) removing the protecting group Is in accordance with the general solid-phase peptide synthesis method of repeating the step, the synthetic peptide to a linear (straight chain) form of peptide - a third step of obtaining a resin connecting body; A sulfide group forming reagent such as DMF / anisole solution, Tl (tfa) 3 (thalium trifluoroacetate) mixture, iodine, Hg (mercury) divalent ions or Ag (silver) monovalent ions was added to the linker to form a resin phase. A fourth step of subjecting to a cyclization reaction by forming a disulfide bridge; Adding a weakly acidic cleavage solution such as trifluoroacetic acid (TFA): triisopropylsilane (TIS) mixed solution or dichloromethane (DCM) to the linker to remove the side chain protecting group and simultaneously cutting the resin from the resin. Somatostatin of the present invention can be prepared through. The PG means a protecting group.
(반응식 2)(Scheme 2)
상기 반응식 2는 옥트레오타이드의 합성에 대한 구체적인 예이다. 상기 반응식 2에서 보는 바와 같이 옥트레오타이드는 소마토스타틴과 같이 이황화 브리지(disulfide bridge)를 포함하고 있으나, 시스테인의 위치가 소마토스타틴과는 달라 반응식 1 보다는 좀 더 복잡한 상기 반응식 2의 방법을 통해 얻을 수 있다. 반응식 2는 시스테인의 측쇄를 t-부틸 계열의 보호기가 수지에 고정된 것을 사용한 예이며, 보호기 제거 조건은 하기 참고예 1에 나타내었다. 보호기가 붙어있는 펩타이드를 각종 탈보호제를 처리한 후 침전, 여과 및 원심분리 등을 거친 후 HPLC로 분리하여 반응속도를 측정한다.Scheme 2 is a specific example for the synthesis of octreotide. As shown in Scheme 2, octreotide contains a disulfide bridge like somatostatin, but the position of cysteine can be obtained through the method of Scheme 2, which is more complicated than Scheme 1, unlike somatostatin. Scheme 2 is an example in which a t-butyl-based protecting group is fixed to the resin using a side chain of cysteine, and the protecting group removing conditions are shown in Reference Example 1 below. Peptides with protecting groups are treated with various deprotecting agents, followed by precipitation, filtration and centrifugation, and then separated by HPLC to measure the reaction rate.
구체적으로 본 발명은 상기 반응식 2에서 보는 바와 같이, A 및 B 방법으로 본원 발명의 옥트레오티드를 제조할 수 있는 데, 먼저, 설프릴기(-SH)를 갖는 수지를 이용하는 A 방법으로서, 본 발명은 브로모왕 수지와 같은 수지에 3-메틸-3-부텐-1-올을 반응시킨 후, 아민 말단이 Fmoc (9-fluorenylmethoxycarbonyl) 기로 보호된 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(SH)-OH 등과 같은 시스테인 유도체를 반응시켜 설피드(-S-)를 갖는 수지를 제조하는 제 1단계; 상기 수지에 트레오니놀 아세탈(threoninol acetal)을 DIC(diisopropyl carbodiimide) 또는 HOBt(N-hydroxylbenzotriazole) 등의 시약 하에서 반응시켜 아미노산이 결합된 수지를 제조하는 제 2단계; 상기 단계의 활성형 수지에 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA (dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 제 3단계 ; 상기 탈보호화된 수지에 아민 말단 및 측쇄가 모두 보호기로 보호된 아미노산 유도체들인 Fmoc-O-tert-butyl-L-threoneinol , Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -DTrp(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH, Boc-DPhe-OH을 DIC (diisopropyl carbodiimide) 또는 HOBt(N-hydroxylbenzotriazole)와 같은 반응시약으로 순차적으로 첨가한 후, 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA (dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 단계를 반복하는 일반적인 고체상 펩타이드 합성법에 따라, 펩타이드를 합성하여 직쇄(straight chain) 형태의 펩타이드-수지 연결체를 얻는 제 4단계 ; 상기 연결체에 DMF/아니솔 용액, Tl(tfa)3 (thalium trifluoroacetate) 혼합액, 요오드, Hg(수은) 2가 이온 또는 Ag(은) 1가 이온 등의 설피드기 형성 시약을 첨가하여 수지상에서 이황화 브리지 (disulfide bridge) 형성을 통한 고리화 반응을 시키는 제 5단계 ; 상기 연결체에 TFA(trifluoroacetic acid): TIS(triisopropylsilane) 혼합수용액 또는 DCM(dichloromethane) 등의 약산성 분해 용액을 첨가하여 측쇄 보호기를 제거함과 동시에 수지로부터 절단하는 제 6단계 를 포함하는 공정을 통하여 본 발명의 옥트레오타이드를 제조가능하다.Specifically, in the present invention, as shown in Scheme 2, the octreotide of the present invention can be prepared by the method A and B. First, as an A method using a resin having a sulfyl group (-SH), invention is then reacted with the resin 3-methyl-3-buten-1-ol in like bromo mowang resin, an amine-terminal Fmoc (9-fluorenylmethoxycarbonyl) of Fmoc (9-fluorenylmethoxycarbonyl) protected with -Cys (SH) -OH A first step of preparing a resin having sulfide (-S-) by reacting a cysteine derivative such as the like; A second step of reacting the resin with threoninol acetal under a reagent such as diisopropyl carbodiimide (DIC) or HOBt (N-hydroxylbenzotriazole) to prepare an amino acid-bonded resin; Fmoc (9-fluorenylmethoxycarbonyl) protecting group is removed by a deprotection reaction which is treated with a reaction solvent such as piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) in the active resin of the step. Third step to do ; Fmoc-O-tert-butyl-L-threoneinol , Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH, Fmoc (9-fluorenylmethoxycarbonyl ) -Thr (tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -DTrp (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc ( 9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH and Boc-DPhe-OH were added sequentially with a reaction reagent such as DIC (diisopropyl carbodiimide) or HOBt (N-hydroxylbenzotriazole), followed by piperidine / dimethyl formamide (DMF). Peptide was synthesized according to the general solid phase peptide synthesis method of repeating the step of removing the Fmoc (9-fluorenylmethoxycarbonyl) protecting group by a deprotecting group reaction treated with a reaction solvent such as (20:80, v / v) or DMA (dimethylacetamide). A fourth step of obtaining a peptide-resin linker in the form of a straight chain; The linkage was added to a sulfide group forming reagent such as DMF / anisole solution, Tl (tfa) 3 (thalium trifluoroacetate) mixture, iodine, Hg (mercury) divalent ions, or Ag (silver) monovalent ions. A fifth step of subjecting to a cyclization reaction by forming a disulfide bridge; The present invention through a process comprising a sixth step of cutting off from the resin at the same time to remove the side chain protecting group by adding a weak acid decomposition solution such as trifluoroacetic acid (TFA): triisopropylsilane (TIS) mixed solution or dichloromethane (DCM) to the linker Octreotide can be prepared.
상기 A 방법과 호환적인 방법이며 본 발명의 옥트레오타이드를 얻기 위한 디설프릴기(-S-S-)를 갖는 수지를 이용하는 B방법으로서, 본 발명은 브로모 왕 (Bromo wang) 수지와 같은 수지에 3-메틸-3-부텐-1-올을 반응 후, KSH(potassium hydrogen sulfide)와 같은 설프화제를 반응시킨 후에 얻어진 설프릴기(SH)를 갖는 수지에 아민 말단이 Fmoc (9-fluorenylmethoxycarbonyl) 기로 보호된 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(SH)-OH 등과 같은 시스테인 유도체를 반응시켜 디설피드(-S-S-)를 갖는 수지를 제조하는 제 1단계; 상기 수지에 트레오니놀 아세탈(threoninol acetal)을 DIC(diisopropyl carbodiimide) 또는 HOBt (N-hydroxylbenzotriazole) 등의 시약하에서 반응시켜 아미노산이 결합된 수지를 제조하는 제 2단계; 상기 단계의 활성형 수지에 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA(dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 제 3단계; 상기 탈보호화된 수지에 아민 말단 및 측쇄가 모두 보호기로 보호된 아미노산 유도체들인 Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -DTrp(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Cys(St-Bu)-OH, Boc-DPhe-OH을 DIC(diisopropyl carbodiimide) 또는 HOBt (N-hydroxylbenzotriazole)와 같은 반응시약으로 순차적으로 첨가한 후, 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA(dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 단계를 반복하는 일반적인 고체상 펩타이드 합성법에 따라, 펩타이드를 합성하여 직쇄 형태의 펩타이드-수지 연결체를 얻는 제 4단계; 상기 연결체에 DMF/아니솔 용액, Tl(tfa)3 (thalium trifluoroacetate) 혼합액, 요오드, Hg(수은) 2가 이온 또는 Ag(은) 1가 이온 등의 설피드기 형성시약을 첨가하여 수지 상에서 이황화 브리지(disulfide bridge) 형성을 통한 고리화 반응을 시키는 제 5단계; 상기 연결체에 TFA(trifluoroacetic acid): TIS(triisopropylsilane) 혼합수용액 또는 DCM(dichloromethane) 등의 약산성 분해 용액을 첨가하여 측쇄 보호기를 제거함과 동시에 수지로부터 절단하는 제 6단계를 포함하는 공정을 통하여 본 발명의 옥트레오타이드를 제조가능하다.
Method B, which is compatible with the method A and uses a resin having a disulfyl group (-SS-) for obtaining the octreotide of the present invention, the present invention is directed to a resin such as Bromo wang resin. After the reaction of -methyl-3-buten-1-ol, the sulfide group (SH) obtained after the reaction of a sulfating agent such as potassium hydrogen sulfide (KSH ) is protected with an amine-terminated Fmoc (9-fluorenylmethoxycarbonyl) group. A first step of preparing a resin having a disulfide (-SS-) by reacting a cysteine derivative such as Fmoc (9-fluorenylmethoxycarbonyl) -Cys (SH) -OH; A second step of reacting the resin with threoninol acetal under a reagent such as diisopropyl carbodiimide (DIC) or HOBt (N-hydroxylbenzotriazole) to prepare an amino acid-bonded resin; Fmoc (9-fluorenylmethoxycarbonyl) protecting group is removed by a deprotection reaction which is treated with a reaction solvent such as piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) in the active resin of the step. A third step of doing; Amino acid derivatives in which both the amine terminal and the side chain are protected with a protecting group in the deprotected resin are Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9 -fluorenylmethoxycarbonyl) -DTrp (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc (9-fluorenylmethoxycarbonyl) -Cys (St-Bu) -OH, Boc-DPhe-OH as diisopropyl carbodiimide (DIC) or Deprotector reaction which is sequentially added with a reaction reagent such as HOBt (N-hydroxylbenzotriazole) and then treated with a reaction solvent such as piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) A fourth step of synthesizing the peptide to obtain a straight-chain peptide-resin linkage according to a general solid-phase peptide synthesis method of repeating the step of removing the Fmoc (9-fluorenylmethoxycarbonyl) protecting group; A sulfide group forming reagent such as DMF / anisole solution, Tl (tfa) 3 (thalium trifluoroacetate) mixture, iodine, Hg (mercury) divalent ions or Ag (silver) monovalent ions was added to the linker to form a resin phase. A fifth step of subjecting to a cyclization reaction by forming a disulfide bridge; The present invention through a process comprising a sixth step of removing the side chain protecting group by adding a weakly acidic decomposition solution such as trifluoroacetic acid (TFA): triisopropylsilane (TIS) mixed solution or dichloromethane (DCM) to the linker, and simultaneously cutting the side chain protecting group. Octreotide can be prepared.
(반응식 3)(Scheme 3)
상기 반응식 3은 시스테인의 측쇄를 트리틸 계열의 보호기가 수지에 고정된 것을 사용한 예이다.Scheme 3 is an example in which a trityl-based protecting group is fixed to the resin by the side chain of cysteine.
구체적으로 본 발명은 상기 반응식 3에서 보는 바와 같은 C방법으로서, 본 발명은 아민 말단이 Fmoc (9-fluorenylmethoxycarbonyl) 기로 보호된 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(SH)-OAct 등과 같은 시스테인 유도체를 2-클로로트리틸 클로라이드 수지 등의 수지와 DIEA(N,N-didisopropylethylamine) 등의 용매하에서 결합시켜 설피드(-S-)를 갖는 수지를 제조하는 제 1단계; 상기 수지에 트레오니놀 아세탈(threoninol acetal)을 DIC (Diisopropyl carbodiimide) 또는 HOBt(N-hydroxylbenzotriazole) 등의 시약하에서 반응시켜 아미노산이 결합된 수지를 제조하는 제 2단계; 상기 단계의 활성형 수지에 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA(dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 제 3단계; 상기 탈보호화된 수지에 아민 말단 및 측쇄가 모두 보호기로 보호된 아미노산 유도체들인 Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -DTrp(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Trt)-OH, Boc-DPhe-OH을 DIC(Diisopropyl carbodiimide) 및 HOBt(N-hydroxylbenzotriazole)와 같은 반응시약으로 순차적으로 첨가한 후, 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA(dimethylacetamide) 등의 반응용매로 처리하는 탈보호기 반응으로 Fmoc (9-fluorenylmethoxycarbonyl) 보호기를 제거하는 단계를 반복하는 일반적인 고체상 펩타이드 합성법에 따라, 펩타이드를 합성하여 직쇄 형태의 펩타이드-수지 연결체를 얻는 제 4단계; 상기 연결체에 DMF/아니솔 용액, Tl(tfa)3(thalium trifluoroacetate) 혼합액, 요오드, Hg(수은) 2가 이온 또는 Ag(은) 1가 이온 등의 설피드기 형성시약을 첨가하여 수지 상에서 이황화 브리지 형성을 통한 고리화 반응을 시키는 제 5단계; 상기 연결체에 TFA(trifluoroacetic acid): TIS(triisopropylsilane) 혼합수용액 또는 DCM(dichloromethane) 등의 약산성 분해 용액을 첨가하여 측쇄 보호기를 제거함과 동시에 수지로부터 절단하는 제 6단계를 포함하는 공정을 통하여 본 발명의 옥트레오타이드를 제조가능하다.Specifically, as the method C, as the present invention is shown in Reaction Scheme 3, the present invention is 2 to cysteine derivatives such as amine-terminal Fmoc (9-fluorenylmethoxycarbonyl) protecting group of Fmoc (9-fluorenylmethoxycarbonyl) -Cys ( SH) -OAct A first step of preparing a resin having sulfide (-S-) by combining a resin such as chlorotrityl chloride resin and a solvent such as DIEA (N, N-didisopropylethylamine); A second step of reacting the resin with threoninol acetal under a reagent such as diisopropyl carbodiimide (DIC) or HOBt (N-hydroxylbenzotriazole) to prepare an amino acid-bonded resin; Fmoc (9-fluorenylmethoxycarbonyl) protecting group is removed by a deprotection reaction which is treated with a reaction solvent such as piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) in the active resin of the step. A third step of doing; Amino acid derivatives in which both the amine terminal and the side chain are protected with a protecting group in the deprotected resin are Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9 -fluorenylmethoxycarbonyl) -DTrp (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Trt) -OH, Boc-DPhe-OH are diisopropyl carbodiimide (DIC) and HOBt ( After adding sequentially with a reaction reagent such as N-hydroxylbenzotriazole), Fmoc is a deprotection group reaction treated with a reaction solvent such as piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide). A fourth step of synthesizing the peptide to obtain a straight-chain peptide-resin linkage according to a general solid-phase peptide synthesis method of repeating the step of removing the (9-fluorenylmethoxycarbonyl) protecting group; A sulfide group forming reagent such as DMF / anisole solution, Tl (tfa) 3 (thalium trifluoroacetate) mixture, iodine, Hg (mercury) divalent ions or Ag (silver) monovalent ions was added to the linker to form a resin phase. A fifth step of subjecting the cyclization reaction to form a disulfide bridge; The present invention through a process comprising a sixth step of removing the side chain protecting group by adding a weakly acidic decomposition solution such as trifluoroacetic acid (TFA): triisopropylsilane (TIS) mixed solution or dichloromethane (DCM) to the linker, and simultaneously cutting the side chain protecting group. Octreotide can be prepared.
상기 반응식 1 내지 3에서 나타내는 바와 같이, 아민 말단이 Fmoc (9-fluorenylmethoxycarbonyl) 기로 보호된 아미노산 유도체들을 사용하게 되는데 Fmoc (9-fluorenylmethoxycarbonyl) 기는 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 또는 DMA (dimethylacetamide)로 처리하여 제거함을 알 수 있다.As shown in Schemes 1 to 3, amino acid derivatives whose amine ends are protected with Fmoc (9-fluorenylmethoxycarbonyl) groups are used. The Fmoc (9-fluorenylmethoxycarbonyl) group is piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) to remove it.
합성된 펩타이드 수지는 도입된 기능기의 종류에 따라 처리 방법이 달라지는데, 하기 표 1 (참고예 1 참조)에 그 경향성을 나타내었다. 최종적으로 얻은 펩타이드는 역상 컬럼 및 이온교환 수지를 이용하여 정제된 형태로 얻어진다. The synthesized peptide resin has a different treatment method depending on the type of functional group introduced, and the tendency is shown in Table 1 (see Reference Example 1). The finally obtained peptide is obtained in purified form using a reversed phase column and an ion exchange resin.
상기 반응식 1 내지 3에서의 펩타이드 합성은 하기 문헌에 기재된 일반적으로 알려진 펩타이드 합성법을 이용할 수 있다(Synthetic Peptides : A User's Guide, G.R. Grant, ed., Freeman & Co.,1992, pp.77-183). 아미노산의 커플링은 각 아미노산의 활성에스테르를 이용하거나 DCC(dicyclohexyl carbodiimide), DIC (diisopropyl carbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluoro- phosphate), PyBOP(Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), HBTU(O-Benzotriazole-N,N,N',N'-tetramethyl- uronium hexafluorophosphate), TBTU(O-(Benzotriazole-1-yl)-N,N,N',N'- tetramethyluronium tetrafluroborate), HATU(2-(1H-7-Azabenzotriaol-1- yl)-1,1,3,3-tetramethyluronium hexfluorophospate), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium ttrafluoro- borate) 및 CDI(carbonyl diimidazole)으로 구성된 군으로부터 선택적으로 사용될 수 있으며, 바람직하게는 DIC(diisopropyl carbodiimide)이 사용가능하다. 그 사용량은 아미노산 당량에 대해 약 1 내지 10 당량, 바람직하게는 1.5 내지 3 당량이 사용된다. 아미노산 유도체의 양은 수지의 치환율에 대해 약 1 내지 10 당량, 바람직하게는 1.5 내지 3 당량이 사용가능하다.Peptide synthesis in Schemes 1 to 3 can use the commonly known peptide synthesis described in the literature ( Synthetic Peptides : A User's Guide, GR Grant, ed., Freeman & Co., 1992, pp. 77-183). Coupling of amino acids is carried out using active esters of each amino acid or by using dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), benzotriazole-1-yl-oxy-tris- (dimethylamino) -phosphonium hexafluoro-phosphate (BOP), and benzotriazole (PyBOP). -1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), O-Benzotriazole-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU), TBTU (O- (Benzotriazole-1-yl) -N, N, N', N'- tetramethyluronium tetrafluroborate), HATU (2- (1H-7-Azabenzotriaol-1-yl) -1,1,3,3-tetramethyluronium hexfluorophospate), TATU (2- (1H-7-Azabenzotriazol-1-yl) It can optionally be used from the group consisting of -1,1,3,3-tetramethyluronium ttrafluoroborate (CDI) and carbonyl diimidazole (CDI), preferably diisopropyl carbodiimide (DIC) can be used. The amount used is about 1 to 10 equivalents, preferably 1.5 to 3 equivalents based on the amino acid equivalent. The amount of the amino acid derivative may be used in an amount of about 1 to 10 equivalents, preferably 1.5 to 3 equivalents, based on the substitution rate of the resin.
상기 아미노산 활성에스테르는 아미노산 유도체의 대칭 무수화물(symmetric anhydride), 혼합 무수화물(mixed anhydride), 펜타플루오르 페닐 에스테르 등 아미노산의 카르복시 말단을 활성화시킨 물질로서 고분자 수지에 결합된 아미노산 당량에 대해 약 1 내지 10 당량 사용하는 것이 바람직하다.The amino acid active ester is a substance that activates the carboxy terminus of an amino acid such as symmetric anhydride, mixed anhydride, pentafluoro phenyl ester of an amino acid derivative, and about 1 to about amino acid equivalents bound to the polymer resin. It is preferable to use 10 equivalents.
본원에 개시된 아민 말단 및 측쇄가 모두 보호된 아미노산 유도체들을 고분자 지지체와 순차적으로 결합시켜 펩타이드를 제조하는 방법으로 기존의 공지된 기술들과는 달리 측쇄가 보호된 형태의 아미노산 유도체들을 사용함으로서, 소마토스타틴 및 그 유사체인 옥트레오타이드 등의 펩타이드를 고순도 및 고수율로 제조할 수 있다.Somatostatin and the like by using amino acid derivatives in the form of protected side chains, unlike the known techniques, to prepare peptides by sequentially combining amino acid derivatives protected with both amine terminal and side chains disclosed herein with a polymer support. Peptides such as chain octreotide can be prepared in high purity and high yield.
본 발명의 고체상에서 소마토스타틴 또는 그 유사체인 옥트레오타이드의 제조방법에 관한 것으로, 개질된 폴리스티렌 수지에 아미노산 유도체들을 순차적으로 반응시켜 펩타이드 수지를 얻고 이 수지에서 펩타이드를 최종적으로 유리시켜 펩타이드를 제조하는 방법으로, 고체상 지지체 상에서 펩타이드를 유리시킴과 동시에 고리화 반응을 수행할 수 있어 용액상 합성법보다 간편하게 할 수 있으며, 종래기술상의 문제점인 고비용 및 소량 생산 등의 단점을 개선하여 목적물질을 고수율 및 저비용으로 대량 생산이 가능하도록 하였다.The present invention relates to a method for preparing somatostatin or an analog of octreotide in the solid phase of the present invention, wherein a peptide resin is obtained by sequentially reacting an amino acid derivative with a modified polystyrene resin to finally release a peptide from the resin to prepare a peptide. By releasing the peptide on the solid phase support and simultaneously carrying out a cyclization reaction, it is easier to carry out than the solution phase synthesis method, and improves the disadvantages of high cost and small amount production, which are the problems of the prior art, to obtain the target material in high yield and low cost. To enable mass production.
이하, 본 발명은 하기 참고예 및 실시예에 의거하여 좀 더 상세하게 설명하고자 한다. Hereinafter, the present invention will be described in more detail based on the following Reference Examples and Examples.
단, 하기 참고예 및 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 여기에 제한되거나 한정되고자 함은 아니다.
However, the following Reference Examples and Examples are only for illustrating the present invention, but the scope of the present invention is not intended to be limited or limited thereto.
참고예Reference Example 1. 시스테인 1. Cysteine 측쇄보호기에Side chain protector 따른 반응결과 Result of reaction
하기 실시예 1 내지 실시예 4에서 사용되는 합성된 펩타이드 수지는 도입된 기능기의 종류에 따라 처리 방법이 달라지는데, 수지상의 시스테인 측쇄 보호기로는 다양한 것이 사용가능하므로 다양한 보호기 사용 및 그에 따른 용매조건에 따른 반응속도를 문헌의 방법들 (Tamamura, H. et al. Int . J. Pept . Pro . Res. 45, 321, 1995; Albericio, F. et al . In "Fmoc Solid Phase Synthesis: A Practical Approach", Chan, W.C. & White, P.D.(Eds.), Oxford Univ. Press, Oxford, 2000, 104; Beekman, N.J.C.M. et al. J. Pept . Res. 50, 357, 1997; Albericio, F. et al. Int . J. Pept . Pro . Res. 37, 402, 1991; Ranganathan, S & Jayaraman, N. J. Chem . Soc ., Chem . Commun . 934, 1991)을 이용하여 하기와 같이 실험하여 표 1에 나타내었다. The synthesized peptide resins used in Examples 1 to 4 may be treated according to the type of functional group introduced, and various resin groups may be used as cysteine side chain protecting groups, so that various protecting groups may be used and solvent conditions thereof. Reaction rate according to the methods of literature (Tamamura, H. et. al . Int . J. Pept . Pro . Res . 45 , 321, 1995; Albericio, F. et al . In "Fmoc Solid Phase Synthesis: A Practical Approach", Chan, WC & White, PD (Eds.), Oxford Univ. Press, Oxford, 2000, 104; Beekman, NJCM et al . J. Pept . Res . 50 , 357, 1997; Albericio, F. et al . Int . J. Pept . Pro . Res . 37 , 402, 1991; Ranganathan, S & Jayaraman, N. J. Chem . Soc , Chem . Commun . 934, 1991) is shown in Table 1 by the experiment as follows.
1-1. 수은 2가 이온(1-1. Mercury divalent ions ( HgHg IIII )을 사용한 Using) 아세타미도메틸(Acm)기Acetamidomethyl (Acm) group 제거 remove
측쇄가 Acm기로 보호된 펩타이드를 10% 초산 수용액에 녹이고(5~10 mg/mL), 빙초산으로 pH 4.0으로 조정하였다. Acm기에 대해 10당량의 Hg(OAc)2를 넣고 초산 또는 암모니아 수용액으로 pH 4.0으로 다시 맞추었다. 용액은 질소 분위기, 상온에서 천천히 교반하였다. Acm기에 대해 20당량의 베타-메르캅토에탄올(β-mercaptoethanol)을 넣고 5시간동안 방치하였다. 원심분리하여 침전을 제거하고 HPLC를 이용하여 상등액에서 염을 제거하였다. Peptides whose side chains were protected with Acm groups were dissolved in 10% aqueous acetic acid solution (5-10 mg / mL) and adjusted to pH 4.0 with glacial acetic acid. 10 equivalents of Hg (OAc) 2 was added to the Acm group and adjusted to pH 4.0 with acetic acid or aqueous ammonia solution. The solution was slowly stirred under nitrogen atmosphere at room temperature. 20 equivalents of beta-mercaptoethanol (β-mercaptoethanol) were added to the Acm group, and left for 5 hours. The precipitate was removed by centrifugation and salts were removed from the supernatant using HPLC.
1-2. 수은 2가 이온(1-2. Mercury divalent ions ( HgHg IIII )을 사용한 Using) 티부틸(tBu)기Tbutyl (tBu) group 제거 remove
측쇄가 tBu기로 보호된 펩타이드를 얼음으로 냉각한 티플루로아세트산(TFA)에 녹이고(5~10 mg/mL), tBu기에 대해 10당량의 Hg(OAc)2를 넣고 질소 분위기, 상온에서 3시간 동안 천천히 교반하였다. 상온에서 감압하여 TFA를 제거하고 잔유물을 10% 초산 수용액에 다시 녹였다. tBu기에 대해 20당량의 베타-메르캅토에탄올(β-mercaptoethanol)을 넣고 5시간동안 방치하였다. 원심분리하여 침전을 제거하고 HPLC를 이용하여 상등액에서 염을 제거하였다. The side chain-protected peptide was dissolved in tfluroacetic acid (TFA) cooled with ice (5-10 mg / mL), and 10 equivalents of Hg (OAc) 2 was added to the tBu group, followed by nitrogen atmosphere at room temperature for 3 hours. Stirred slowly. TFA was removed under reduced pressure at room temperature, and the residue was dissolved in 10% acetic acid aqueous solution. 20 equivalents of beta-mercaptoethanol (β-mercaptoethanol) were added to the tBu group and left for 5 hours. The precipitate was removed by centrifugation and salts were removed from the supernatant using HPLC.
1-3. 은 1가 이온(1-3. Silver monovalent ions ( AgAg I)을 사용한 Using i) 아세타미도메틸(Acm)기Acetamidomethyl (Acm) group 제거 remove
측쇄가 Acm기로 보호된 펩타이드를 TFA/아니솔(99:1, v/v)에 녹였다(1 mg/mL). Acm기에 대해 100당량의 은 트리플로아세테이트(Ag trifluoroacetate)를 넣고 4℃에서 2시간 동안 교반하였다. 에테르를 가해 은염(silver salt) 형태의 펩타이드를 얻고 원심분리하여 회수하였다. 환원된 형태의 펩타이드를 얻고자 할 경우, 1M 초산 용액에서 Acm기에 대해 40당량의 DTT(dithiothreitol)를 가하고 25℃에서 3시간 처리한다. 원심분리하여 침전을 제거하고 HPLC를 이용하여 상등액에서 염을 제거하였다. 산화된 형태의 펩타이드를 얻고자 할 경우, 상온에서 염산 수용액/DMSO(1:1, v/v)로 처리한 후 여과하여 AgCl을 제거하고 HPLC를 이용하여 분리하였다.Peptides with side chain protected Acm groups were dissolved in TFA / Anisole (99: 1, v / v) (1 mg / mL). 100 equivalents of silver trifluoroacetate (Ag trifluoroacetate) was added to the Acm group, followed by stirring at 4 ° C for 2 hours. Ether was added to obtain a silver salt peptide and recovered by centrifugation. In order to obtain the reduced form of the peptide, 40 equivalents of DTT (dithiothreitol) is added to the Acm group in 1M acetic acid solution and treated at 25 ° C. for 3 hours. The precipitate was removed by centrifugation and salts were removed from the supernatant using HPLC. To obtain the oxidized form of the peptide, the mixture was treated with aqueous hydrochloric acid / DMSO (1: 1, v / v) at room temperature, filtered to remove AgCl, and separated using HPLC.
1-4. 1-4. 티올(thiol)을Thiol 사용한 Used 티부틸티오(StBU)기Thibutylthio (StBU) group 제거 remove
측쇄가 StBU기로 보호된 펩타이드를 최소량의 0.1 M 탄화수소 암모늄 (ammonium bicarbonate)에 녹였다. 잘 녹지 않을 경우 DMF 또는 MeCN을 첨가할 수도 있다. 아르곤하에서 5분동안 교반하여 용액에서 공기(특히 산소)를 제거하였다. 20당량의 DTT/0.1 M 탄화수소 암모늄 (ammonium bicarbonate)를 넣고 아르곤하에서 2시간 동안 교반하였다. 초산으로 pH 2로 조정하고 바로 HPLC로 정제하였다. 참고로, Cys(StBu)는 고체상에서 펩타이드가 결합된 수지를 베타-메르캅토에탄올 (β- mercaptoethanol)/DMF (1:1, v/v)로 상온에서 5시간동안 처리하면 탈보호되었다.Peptides whose side chains were protected with StBU groups were dissolved in a minimum amount of 0.1 M ammonium bicarbonate. If it does not melt well, you can add DMF or MeCN. Stir under argon for 5 minutes to remove air (especially oxygen) from the solution. 20 equivalents of DTT / 0.1 M ammonium bicarbonate was added and stirred under argon for 2 hours. Adjusted to pH 2 with acetic acid and immediately purified by HPLC. For reference, Cys (StBu) was deprotected when the peptide-bound resin was treated with beta-mercaptoethanol / DMF (1: 1, v / v) at room temperature for 5 hours.
1-5. 포스핀(1-5. Phosphine ( phosphinephosphine )을 사용한 Using) 티부틸티오(StBU)기Thibutylthio (StBU) group 제거 remove
측쇄가 StBU기로 보호된 펩타이드를 pH 7.8 암모늄 아세테이트 완충액(ammonium acetate buffer)/n-프로파놀(n-propanol)(1:1, v/v)에 녹였다. 아르곤하에서 5분동안 교반하여 용액에서 공기(특히 산소)를 제거하였다. 100당량의 0.6 M 트리-n-부틸포스핀(tri-n-butylphosphine)/n-프로파놀(n-propanol)을 가하고 아르곤하에서 30분동안 교반하였다. 감압하여 완전히 건조하고 탈기된(digassed) 완충용액에 다시 녹인 후 연과하고 HPLC로 정제하였다.Peptides with side chain protected StBU groups were dissolved in pH 7.8 ammonium acetate buffer / n-propanol (1: 1, v / v). Stir under argon for 5 minutes to remove air (especially oxygen) from the solution. 100 equivalents of 0.6 M tri-n-butylphosphine / n-propanol were added and stirred under argon for 30 minutes. It was completely dried under reduced pressure, re-dissolved in a digassed buffer solution, filtered and purified by HPLC.
상기 기능기의 종류에 따른 반응속도를 요약한 표 1에서 +는 반응이 잘 진행됨을, -는 그 반대를 뜻하며, n은 조사가 충분히 이루어지지 않았음을 나타낸다.
In Table 1, which summarizes the reaction rate according to the type of the functional group, + means that the reaction proceeds well,-means the opposite, and n indicates that the irradiation was not sufficiently performed.
2. 반응 생성물은 disulfide bridge을 갖고 있음.
3. TFA(trifluoroacetic acid), TFMSA(trifluoromethanesulfonic acid), Acm(acetamidomethyl), Tacm(trimethylacetamidomethyl), Trt(triphenylmethyl; trityl), Hg(수은), Ag(은), Tl(thalium), I2(요오드), tBu(tert-butyl), StBu(tert-butylthio)(Ref.) 1. The reaction should proceed in TFA.
2. The reaction product has a disulfide bridge.
3.TFA (trifluoroacetic acid), TFMSA (trifluoromethanesulfonic acid), Acm (acetamidomethyl), Tacm (trimethylacetamidomethyl), Trt (triphenylmethyl; trityl), Hg (mercury), Ag (silver), Tl (thalium), I 2 (iodine) ), t Bu (tert-butyl), S t Bu (tert-butylthio)
실시예Example 1. One. 소마토스타틴(Somatostatin)의Somatostatin 제조 Produce
1-1. 1-1. Fmoc(9-fluorenylmethoxycarbonyl)Fmoc (9-fluorenylmethoxycarbonyl) -Cys(Acm)-CTR-Cys (Acm) -CTR
유리 필터가 부착된 반응기(주문제작품, KC Scientific, 한국)에 2-CTCR(2-chlorotrityl chloride resin; PRSC5055, 비드테크, 한국) 1 g(1.2 mmol/g, 1.2 mmol)을 넣고 디클로로메탄 15 mL을 넣어 팽윤시켰다. Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH 750 mg(1.8 mmol)을 넣고 녹인 후 DIEA(N,N-diisopropylethylamine) 313 ㎕(1.8 mmol)을 넣어 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH와 2-CTCR을 결합시켰다. 수지는 무수 메탄올, 디메틸포름아미드, 디클로메탄으로 세척하였다. Fmoc (9-fluorenylmethoxycarbonyl) 적정 결과 수지는 0.6 mmol/g의 치환율을 보였다.1 g (1.2 mmol / g, 1.2 mmol) of 2-CTCR (2-chlorotrityl chloride resin; PRSC5055, Bidtech, Korea) was added to a reactor equipped with a glass filter (custom product, KC Scientific, Korea), and 15 mL of dichloromethane. To swell. Dissolve with 750 mg (1.8 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH, and add 313 μl (1.8 mmol) of DIEA (N, N-diisopropylethylamine) to Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH and 2-CTCR were bound. The resin was washed with anhydrous methanol, dimethylformamide and dichloromethane. Fmoc (9-fluorenylmethoxycarbonyl) titration showed that the resin had a substitution rate of 0.6 mmol / g.
1-2. 1-2. 펩타이드Peptide (( 소마토스타틴Somatostatin 전구체)- Precursor) CTRCTR
상기 실시예 1-1에서 얻은 수지 1 g 을 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 용액으로 2회 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 을 제거하였다. DMF로 수지를 2회 세척한 후 DMF 10 mL에 팽윤시키고 Fmoc(9-fluorenylmethoxycarbonyl) -Ser(tBu)-OH 345 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg(0.9 mmol)를 넣고 1.5시간동안 반응시켰다. 얻어진 수지를 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 상기와 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH 358 mg (0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg (0.9 mmol)를 사용하여 반응시켰다. 얻어진 수지를 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하였다. 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH 349 mg(0.9 mmol), DIC 139 ㎕ (0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척한 후 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하였다. 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH 358 mg(0.9 mmol), DIC 139 ㎕ (0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하고 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하였다. 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH 422 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg (0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Trp(Boc)-OH 474 mg (0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH 349 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH 349 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하였다. 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Asn(Trt)-OH 537 mg(0.9 mmol), DIC 139 ㎕ (0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH 422 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg (0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하였다. 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH 373 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 Fmoc(9-fluorenylmethoxycarbonyl) -Gly-OH 268 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 얻어진 수지를 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 상기와 같은 방법으로 Boc-Ala-OH 170 mg(0.9 mmol), DIC 139 ㎕(0.9 mmol), HOBt 122 mg(0.9 mmol)를 사용하여 반응시키고 수지를 세척하였다.1 g of the resin obtained in Example 1-1 was treated twice with a piperidine / DMF (dimethyl formamide) (20:80, v / v) solution to remove Fmoc (9-fluorenylmethoxycarbonyl) . Wash the resin twice with DMF and swell in 10 mL of DMF and 345 mg (0.9 mmol) Fmoc (9-fluorenylmethoxycarbonyl) -Ser (tBu) -OH, 139 μl (0.9 mmol) DIC, 122 mg (0.9 mmol) HOBt Was added and reacted for 1.5 hours. The obtained resin was treated with piperidine / DMF (dimethyl formamide) (20:80, v / v) to remove Fmoc (9-fluorenylmethoxycarbonyl) group and Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu)- The reaction was carried out using 358 mg (0.9 mmol) of OH, 139 μl (0.9 mmol) of DIC, and 122 mg (0.9 mmol) of HOBt. The obtained resin was treated with piperidine / DMF (dimethyl formamide) (20:80, v / v) to remove the Fmoc (9-fluorenylmethoxycarbonyl) group. In the same manner, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH 349 mg (0.9 mmol), DIC 139 μl (0.9 mmol) and HOBt 122 mg (0.9 mmol) were reacted and the resin was washed again, followed by piperidine / The Fmoc (9-fluorenylmethoxycarbonyl) group was removed by treatment with dimethyl formamide (20:80, v / v) . Reaction using Fmoc (9-fluorenylmethoxycarbonyl) -Thr ( tBu) -OH 358 mg (0.9 mmol), DIC 139 ㎕ (0.9 mmol), HOBt 122 mg (0.9 mmol) in the same way and the resin washed again and piperidinyl The Fmoc (9-fluorenylmethoxycarbonyl) group was removed by treatment with dine / DMF (dimethyl formamide) (20:80, v / v) . In the same manner, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH 422 mg (0.9 mmol), DIC 139 μl (0.9 mmol) and HOBt 122 mg (0.9 mmol) were reacted and the resin was washed again. Treatment with piperidine / DMF (dimethyl formamide) (20:80, v / v) removes Fmoc (9-fluorenylmethoxycarbonyl) groups and in the same way Fmoc (9-fluorenylmethoxycarbonyl) -Trp (Boc) -OH 474 mg (0.9 mmol), 139 μl (0.9 mmol) of DIC, and 122 mg (0.9 mmol) of HOBt were reacted and the resin was washed again. Treatment with piperidine / DMF (dimethyl formamide) (20:80, v / v) removes Fmoc (9-fluorenylmethoxycarbonyl) groups, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH 349 mg (0.9 mmol), DIC 139 μl (0.9 mmol), 122 mg (0.9 mmol) of HOBt were reacted, and the resin was washed again. Treatment with piperidine / DMF (dimethyl formamide) (20:80, v / v) removes Fmoc (9-fluorenylmethoxycarbonyl) groups, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH 349 mg (0.9 mmol), DIC 139 μl (0.9 mmol), 122 mg (0.9 mmol) of HOBt were reacted, and the resin was washed again. Fmoc (9-fluorenylmethoxycarbonyl) groups were removed by treatment with piperidine / dimethylformamide (DMF) (20:80, v / v) . In the same manner, 537 mg (0.9 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -Asn (Trt) -OH, 139 μl (0.9 mmol) of DIC and 122 mg (0.9 mmol) of HOBt were reacted and the resin was washed again. Treated with piperidine / DMF (dimethyl formamide) (20:80, v / v) to remove the Fmoc (9-fluorenylmethoxycarbonyl) group, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH 422 mg (0.9 mmol), 139 μl (0.9 mmol) of DIC and 122 mg (0.9 mmol) of HOBt were reacted and the resin was washed again. Fmoc (9-fluorenylmethoxycarbonyl) groups were removed by treatment with piperidine / dimethylformamide (DMF) (20:80, v / v) . In the same manner, 373 mg (0.9 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH, 139 μl (0.9 mmol) of DIC, and 122 mg (0.9 mmol) of HOBt were reacted and the resin was washed again. Treated with piperidine / dimethyl formamide (DMF) (20:80, v / v) to remove the Fmoc (9-fluorenylmethoxycarbonyl) group, Fmoc (9-fluorenylmethoxycarbonyl) -Gly-OH 268 mg (0.9 mmol), DIC 139 μl (0.9 mmol), 122 mg (0.9 mmol) of HOBt were reacted, and the resin was washed again. The obtained resin was treated with piperidine / DMF (dimethyl formamide) (20:80, v / v) to remove Fmoc (9-fluorenylmethoxycarbonyl) group, and Boc-Ala-OH 170 mg (0.9 mmol), 139 μl (0.9 mmol) of DIC and 122 mg (0.9 mmol) of HOBt were reacted and the resin was washed.
펩타이드 수지 70 mg에 대해 DMF/아니솔 (19:1, v/v) 용액과 Tl(tfa)3 1.63 g (1.4 mmol)을 넣고 4시간 동안 반응시켰다. 수지를 세척한 후 TFA:TIS:물(90:5:5) 용액으로 처리하여 그 액을 모으고, 수지를 DCM과 메탄올로 세척하여 그 여액까지 같이 모았다. 모아진 용액을 감압 농축시키고 차가운 에테르에 농축액을 한방울씩 적가하여 침전을 유도하고 냉장실에 방치하여 펩타이드 378 mg (수율 55%)을 얻었다.
DMF / anisole (19: 1, v / v) solution and 1.63 g (1.4 mmol) of Tl (tfa) 3 were added to 70 mg of the peptide resin and reacted for 4 hours. The resin was washed and then treated with a TFA: TIS: water (90: 5: 5) solution to collect the liquid, and the resin was washed with DCM and methanol to collect the filtrate together. The combined solution was concentrated under reduced pressure, and the concentrated solution was added dropwise to cold ether dropwise to induce precipitation and left in the refrigerator to obtain 378 mg (55% yield) of the peptide.
실시예Example 2. 2. 옥트레오타이드(Octreotide)의Of octreotide 제조예Production Example (A 방법) (A method)
2-1. 2-1. Fmoc(9-fluorenylmethoxycarbonyl)Fmoc (9-fluorenylmethoxycarbonyl) -Threoninol(tBu)-CTR-Threoninol (tBu) -CTR
유리 필터가 부착된 반응기(주문제작품, KC Scientific, 한국)에 2-CTCR (2-chlorotrityl chloride resin) 1 g (1.2 mmol/g, 1.2 mmol)을 넣고 디클로로메탄 15 mL을 넣어 팽윤시켰다. Fmoc-O-tert-butyl-L-threoneinol 1.38 g (3.6 mmol)을 넣고 녹인 후 DIEA 628 ㎕ (3.6 mmol)을 넣어 Fmoc-O-tert-butyl-L-threoneinol 과 2-CTCR을 결합시켰다. 수지는 메탄올, 디메틸포름아미드, 디클로메탄으로 세척하였다. Fmoc (9-fluorenylmethoxycarbonyl) 적정 결과 수지는 0.44 mmol/g의 치환율을 보였다.1 g (1.2 mmol / g, 1.2 mmol) of 2-CTCR (2-chlorotrityl chloride resin) was added to a reactor equipped with a glass filter (custom product, KC Scientific, Korea) and swelled with 15 mL of dichloromethane. 1.38 g (3.6 mmol) of Fmoc-O-tert-butyl-L-threoneinol was added to dissolve it, and 628 μl (3.6 mmol) of DIEA was added to combine Fmoc-O-tert-butyl-L-threoneinol with 2-CTCR. The resin was washed with methanol, dimethylformamide, dichloromethane. Fmoc (9-fluorenylmethoxycarbonyl) titration showed that the resin had a substitution rate of 0.44 mmol / g.
2-2. 2-2. 페타이드Petide (( 옥트레오타이드Octreotide 전구체)- Precursor) CTRCTR
상기 실시예 2-1에서 얻은 수지 1g 을 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 용액으로 2회 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 을 제거하였다. DMF로 수지를 2회 세척한 후 DMF 10 mL에 팽윤시키고 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH 547 mg (1.32 mmol), DIC 204 ㎕(1.32 mmol), HOBt 178 mg (1.32 mmol)를 넣고 1.5 시간동안 반응시켰다. 얻어진 수지를 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 상기와 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH 525 mg (1.32 mmol), DIC 204 ㎕ (1.32 mmol), HOBt 178 mg (1.32 mmol)를 사용하여 반응시켰다. 얻어진 수지를 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 상기와 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH 618 mg (1.32 mmol), DIC 204 ㎕ (1.32 mmol), HOBt 178 mg (1.32 mmol)를 사용하여 반응시키고 다시 수지를 세척한 후 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하였다. 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -DTrp(Boc)-OH 695 mg (1.32 mmol), DIC 204 ㎕ (1.32 mmol), HOBt 178 mg (1.32 mmol)를 사용하여 반응시키고 다시 수지를 세척하고 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 상기와 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH 511 mg (1.32 mmol), DIC 204 ㎕ (1.32 mmol), HOBt 178 mg (1.32 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 같은 방법으로 Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Acm)-OH 547 mg (1.32 mmol), DIC 204 ㎕ (1.32 mmol), HOBt 178 mg (1.32 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다. 피페리딘/DMF(dimethyl formamide)(20:80, v/v) 으로 처리하여 Fmoc (9-fluorenylmethoxycarbonyl) 기를 제거하고 Boc-DPhe-OH 350 mg (1.32 mmol), DIC 204 ㎕ (1.32 mmol), HOBt 178 mg (1.32 mmol)를 사용하여 반응시키고 다시 수지를 세척하였다.1 g of the resin obtained in Example 2-1 was treated with a piperidine / DMF (dimethyl formamide) (20:80, v / v) solution twice to remove Fmoc (9-fluorenylmethoxycarbonyl) . The resin was washed twice with DMF and then swelled in 10 mL of DMF and 547 mg (1.32 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH, 204 μl (1.32 mmol) of DIC, 178 mg (1.32 mmol) of HOBt Was added and reacted for 1.5 hours. The obtained resin was treated with piperidine / DMF (dimethyl formamide) (20:80, v / v) to remove Fmoc (9-fluorenylmethoxycarbonyl) group and Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu)- The reaction was performed using 525 mg (1.32 mmol) of OH, 204 μl (1.32 mmol) of DIC, and 178 mg (1.32 mmol) of HOBt. The obtained resin was treated with piperidine / DMF (dimethyl formamide) (20:80, v / v) to remove Fmoc (9-fluorenylmethoxycarbonyl) group and Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc)- Reaction with 618 mg (1.32 mmol) OH, 204 μl (1.32 mmol) DIC, 178 mg (1.32 mmol) HOBt and washing the resin again followed by piperidine / dimethyl formamide (DMF) (20:80, v / v) to remove Fmoc (9-fluorenylmethoxycarbonyl) groups. In the same manner, 695 mg (1.32 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -DTrp (Boc) -OH, 204 μl (1.32 mmol) of DIC, and 178 mg (1.32 mmol) of HOBt were reacted, and the resin was washed again and piperied. Dimethyl formamide (20:80, v / v) to remove Fmoc (9-fluorenylmethoxycarbonyl) group, and Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH 511 mg (1.32 mmol), Reaction was carried out using 204 μl (1.32 mmol) of DIC, 178 mg (1.32 mmol) of HOBt, and the resin was washed again. Treatment with piperidine / DMF (dimethyl formamide) (20:80, v / v) removes the Fmoc (9-fluorenylmethoxycarbonyl) group and in the same way Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Acm) -OH 547 mg (1.32) mmol), 204 μl (1.32 mmol) of DIC, 178 mg (1.32 mmol) of HOBt, and the resin were washed again. Treatment with piperidine / dimethyl amide (DMF) (20:80, v / v) removes Fmoc (9-fluorenylmethoxycarbonyl) groups, 350 mg (1.32 mmol) of Boc-DPhe-OH, 204 μl (1.32 mmol) of DIC, Reaction was performed using 178 mg (1.32 mmol) of HOBt and the resin was washed again.
펩타이드 수지 70 mg에 대해 DMF/아니솔 (19:1, v/v) 용액과 Tl(tfa)3 490 mg (0.9 mmol)을 넣고 4시간 동안 반응시켰다. 수지를 세척한 후 TFA:TIS:물 (90:5:5) 용액으로 처리하여 그 액을 모으고, 수지를 DCM과 메탄올로 세척하여 그 여액까지 같이 모았다. 모아진 용액을 감압 농축시키고 차가운 에테르에 농축액을 한방울씩 적가하여 침전을 유도하고 냉장실에 방치하여 펩타이드 245 mg (수율 78%)을 얻었다.
DMF / anisole (19: 1, v / v) solution and 490 mg (0.9 mmol) of Tl (tfa) 3 were added to 70 mg of the peptide resin and reacted for 4 hours. The resin was washed and then treated with a TFA: TIS: water (90: 5: 5) solution to collect the solution, and the resin was washed with DCM and methanol to collect the filtrate together. The combined solution was concentrated under reduced pressure, and the concentrated solution was added dropwise to cold ether dropwise to induce precipitation and left in the refrigerator to obtain 245 mg of peptide (78% yield).
실시예Example 3. 3. 옥트레오타이드(Octreotide)의Of octreotide 제조예Production Example (B 방법) (B method)
브로모 왕 (Bromo wang) 수지 1 g (1.0 mmol/g, 1 mmol)에 3-메틸-3-부텐-1-올 303 ㎕ (3 mmol), DIEA 523 ㎕ (3 mmol)을 넣고 반응시켰다. KSH 3.6 g(50 mmol)을 넣고 DMF 용매하에서 60℃ 에서 반응시킨 후 Fmoc(9-fluorenylmethoxycarbonyl) -Cys-OH 1.7 g (5 mmol)을 넣고 공기를 불어넣어 반응시켰다. Fmoc 적정 결과, 상기 수지는 0.35 mmol/g의 치환율을 보였다.To 1 g (1.0 mmol / g, 1 mmol) of Bromo wang resin, 303 µl (3 mmol) of 3-methyl-3-butene-1-ol and 523 µl (3 mmol) of DIEA were added and reacted. 3.6 g (50 mmol) of KSH was added and reacted at 60 ° C. in a DMF solvent. Then, 1.7 g (5 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -Cys-OH was added thereto and reacted with air. As a result of the Fmoc titration, the resin showed a substitution rate of 0.35 mmol / g.
Fmoc(9-fluorenylmethoxycarbonyl) -트레오닐(threoninol) 3 g(15.7 mmol)과 무수 아세톤을 산 촉매하에서 반응시켜 아세탈을 얻고 정제한 TFA로 10분간 처리하고 정제 에테르를 가해 트레오니놀 아세탈(threoninol acetal) 2.7 g (75%)을 얻었다.3 g (15.7 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -threoninol was reacted with anhydrous acetone under an acid catalyst to obtain acetal, treated with purified TFA for 10 minutes, and purified ether was added to threoninol acetal. 2.7 g (75%) was obtained.
앞에서 얻은 수지에 트레오니놀 아세탈 243 mg(1.05 mmol), DIC 163 ㎕ (1.05 mmol), HOBt 142 mg (1.05 mmol)를 넣고 3시간 동안 반응시켰다. 이후 반응은 상기 실시예 2와 동일한 공정으로 수행하였다. 단, 사용 아미노산 유도체는 Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -DTrp(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Cys(StBu)-OH, Boc-DPhe-OH 였으며 당량 비율은 실시예 2에 준하여 실시하였다.243 mg (1.05 mmol) of threoninol acetal, 163 μl (1.05 mmol) of DIC, and 142 mg (1.05 mmol) of HOBt were added to the obtained resin and reacted for 3 hours. After the reaction was carried out in the same process as in Example 2. The amino acid derivatives used are Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -DTrp (Boc) -OH, Fmoc ( 9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc (9-fluorenylmethoxycarbonyl) -Cys (StBu) -OH, Boc-DPhe-OH, and the equivalent ratio were carried out according to Example 2.
펩타이드 수지 70 mg에 대해 DMF/아니솔 (19:1, v/v) 용액 및 Tl(tfa)3 381 mg (0.7 mmol)을 넣고 4시간 동안 반응시켰다. 수지를 세척한 후 TFA:TIS:물 (90:5:5) 용액으로 처리하여 그 액을 모으고, 수지를 DCM 및 메탄올로 세척하여 그 여액까지 같이 모았다. 모아진 용액을 감압 농축시키고 차가운 에테르에 농축액을 한방울씩 적가하여 침전을 유도하고 냉장실에 방치하여 펩타이드 167 mg (수율 67%)을 얻었다.
DMF / anisole (19: 1, v / v) solution and 381 mg (0.7 mmol) of Tl (tfa) 3 were added to 70 mg of the peptide resin and reacted for 4 hours. The resin was washed and then treated with a TFA: TIS: water (90: 5: 5) solution to collect the liquid, and the resin was washed with DCM and methanol to collect the filtrate together. The combined solution was concentrated under reduced pressure, and the concentrated solution was added dropwise to cold ether dropwise to induce precipitation and left in the refrigerator to obtain 167 mg (yield 67%) of the peptide.
실시예Example 4. 4. 옥트레오타이드Octreotide ( ( OctreotideOctreotide )의 )of 제조예Production Example (C 방법) (C method)
트리틸 수지 (PRSC5055, 비드테크) 1 g (1.2 mmol/g, 1.2 mmol)에 DMF 용매하에서 Fmoc(9-fluorenylmethoxycarbonyl) -Cys-OH 1.24 g(3.6 mmol), DIEA 628 ㎕ (3.6 mmol)을 넣어 반응시켜 2-CTCR와 결합시켰다. Fmoc (9-fluorenylmethoxycarbonyl) 적정 결과, 상기 수지는 0.50 mmol/g의 치환율을 보였다.To 1 g (1.2 mmol / g, 1.2 mmol) of trityl resin (PRSC5055, Beadtech), 1.24 g (3.6 mmol) of Fmoc (9-fluorenylmethoxycarbonyl) -Cys-OH and 628 μl (3.6 mmol) of DIEA were added in a DMF solvent. Reaction was combined with 2-CTCR. Fmoc (9-fluorenylmethoxycarbonyl) titration showed that the resin had a substitution rate of 0.50 mmol / g.
Fmoc(9-fluorenylmethoxycarbonyl) -트레오니놀 (#37201, GL Biochem, 15.7 mmol) 3 g 과 무수 아세톤을 산 촉매하에서 반응시켜 아세탈을 얻고 정제한 TFA로 10분간 처리하고 정제 에테르를 가해 트레오니놀 아세탈 (threoninol acetal)을 2.7 g (수율 75%)얻었다. 3 g of Fmoc (9-fluorenylmethoxycarbonyl) -threoninol (# 37201, GL Biochem, 15.7 mmol) is reacted with anhydrous acetone under an acid catalyst to obtain acetal. The mixture is treated with purified TFA for 10 minutes and purified ether is added to threoninol acetal. 2.7 g (yield 75%) of (threoninol acetal) were obtained.
앞에서 얻은 수지에 트레오니놀 아세탈 347 mg (1.5 mmol), DIC 232 ㎕ (1.5 mmol), HOBt 203 mg (1.5 mmol)를 넣고 3시간동안 반응시켰다. 이후 반응은 실시예 2와 같은 방법으로 진행하였다. 단, 사용 아미노산 유도체는 Fmoc(9-fluorenylmethoxycarbonyl) -Thr(tBu)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Lys(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -DTrp(Boc)-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc(9-fluorenylmethoxycarbonyl) -Cys(Trt)-OH, Boc-DPhe-OH 였으며 당량 비율은 실시예 2에 준하여 실시하였다.347 mg (1.5 mmol) of threoninol acetal, 232 μl (1.5 mmol) of DIC, and 203 mg (1.5 mmol) of HOBt were added to the resin, which was reacted for 3 hours. After the reaction was carried out in the same manner as in Example 2. The amino acid derivatives used are Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -DTrp (Boc) -OH, Fmoc ( 9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Trt) -OH, Boc-DPhe-OH, and the equivalent ratio were carried out according to Example 2.
펩타이드 수지 70 mg에 대해 DMF/아니솔 (19:1, v/v) 용액 및 Tl(tfa)3 1.63 g (3 mmol)을 넣고 4시간 동안 반응시켰다. 수지를 세척한 후 TFA:TIS:물 (90:5:5) 용액으로 처리하여 그 액을 모으고, 수지를 DCM과 메탄올로 세척하여 그 여액까지 같이 모았다. 모아진 용액을 감압 농축시키고 차가운 에테르에 농축액을 한방울씩 적가하여 침전을 유도하고 냉장실에 방치하여 펩타이드 257 mg (수율 72%)을 얻었다.DMF / anisole (19: 1, v / v) solution and 1.63 g (3 mmol) of Tl (tfa) 3 were added to 70 mg of the peptide resin and reacted for 4 hours. The resin was washed and then treated with a TFA: TIS: water (90: 5: 5) solution to collect the solution, and the resin was washed with DCM and methanol to collect the filtrate together. The collected solution was concentrated under reduced pressure, and the concentrated solution was added dropwise to cold ether to induce precipitation and left in the refrigerator to obtain 257 mg of peptide (72% yield).
Claims (3)
A first step of preparing a resin having a sulfide (-S-) by combining a cysteine derivative whose amine end is protected with a Fmoc (9-fluorenylmethoxycarbonyl) group in a solvent with a resin; A second step of preparing a resin having an amino acid bound by reacting threoninol acetal with the resin; The agent for removing the Fmoc (9-fluorenylmethoxycarbonyl) protecting group by a deprotection reaction which is treated with piperidine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) reaction solvent in the active resin of the step Step 3; Amino acid derivatives in which both the amine terminal and the side chain are protected with a protecting group in the deprotected resin are Fmoc (9-fluorenylmethoxycarbonyl) -Thr (tBu) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Lys (Boc) -OH, Fmoc (9 -fluorenylmethoxycarbonyl) -DTrp (Boc) -OH, Fmoc (9-fluorenylmethoxycarbonyl) -Phe-OH, Fmoc (9-fluorenylmethoxycarbonyl) -Cys (Trt) -OH, and then sequentially adding the Boc-DPhe-OH, piperidin According to the solid-phase peptide synthesis method of repeating the step of removing the Fmoc (9-fluorenylmethoxycarbonyl) protecting group by the deprotecting group reaction with a dine / DMF (dimethyl formamide) (20:80, v / v) or DMA (dimethylacetamide) reaction solvent, A fourth step of synthesizing the peptide to obtain a peptide-resin linker in the form of a straight chain; The disulfide bridge on the resin by adding a DMF / anisole solution, Tl (tfa) 3 (thalium trifluoroacetate) mixed solution, iodine, Hg (mercury) divalent ions or Ag (silver) monovalent ions sulfide group forming reagent to the linker a fifth step of causing a cyclization reaction through formation of a disulfide bridge; TFA (trifluoroacetic acid): TIS (triisopropylsilane) mixed solution or DCM (dichloromethane) weakly acidic cleavage solution is added to the linker to remove the side chain protecting group and at the same time prepared through a process comprising a step of cutting from the resin Method for producing octreotide, characterized in that.
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