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WO2020017919A1 - Novel intermediate used for physiologically active polypeptide and method for preparing same - Google Patents

Novel intermediate used for physiologically active polypeptide and method for preparing same Download PDF

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Publication number
WO2020017919A1
WO2020017919A1 PCT/KR2019/008935 KR2019008935W WO2020017919A1 WO 2020017919 A1 WO2020017919 A1 WO 2020017919A1 KR 2019008935 W KR2019008935 W KR 2019008935W WO 2020017919 A1 WO2020017919 A1 WO 2020017919A1
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Prior art keywords
trt
tbu
resin
boc
lys
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PCT/KR2019/008935
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French (fr)
Korean (ko)
Inventor
최원경
김나리
박종환
박성준
김남두
조영범
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한미정밀화학주식회사
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Publication of WO2020017919A1 publication Critical patent/WO2020017919A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F12/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
    • C08F12/02—Monomers containing only one unsaturated aliphatic radical
    • C08F12/04—Monomers containing only one unsaturated aliphatic radical containing one ring
    • C08F12/06—Hydrocarbons
    • C08F12/08—Styrene
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
    • C08G81/02—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers at least one of the polymers being obtained by reactions involving only carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G85/00—General processes for preparing compounds provided for in this subclass
    • 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

Definitions

  • the present invention relates to novel intermediates used in physiologically active polypeptides and methods for their preparation. More specifically, the present invention relates to a method for producing a bioactive polypeptide more safely and efficiently, and to a novel polypeptide intermediate and a method for producing the same.
  • Diabetes-related diseases including obesity and type 2 diabetes, are one of the major metabolic diseases occurring in modern society and are recognized as an important threat to health around the world, and economic costs are increasing rapidly.
  • glucagon derivatives Glucagon is produced in the pancreas when blood sugar begins to drop due to medication or disease, hormone or enzyme deficiency. Glucagon signals the breakdown of glycogen in the liver to release glucose and raises blood sugar levels to normal levels. In addition, glucagon has been reported to exhibit anti-obesity effects by promoting lipolysis by activating hormone-sensitive lipase of appetite suppression and adipocytes in addition to the effect of increasing blood glucose. Various studies are in progress.
  • Korean Patent Laid-Open Publication No. 10-2017-0080521 discloses a triple activator having activity on both glucagon, GLP-1, and GIP receptor and use thereof.
  • Such peptides may be formed from substitution, addition, removal, modification, and combinations thereof of at least one or more amino acids in a native glucagon sequence, and more specifically, in Formula 1
  • An isolated peptide is disclosed comprising the indicated amino acid sequence.
  • the peptides can be prepared according to their length by methods known in the art, such as by synthetic peptide synthesizers, by genetic engineering techniques, or by any other method. In order to use the peptides prepared by such various manufacturing methods as pharmaceuticals, high purity quality, yield suitable for commercialization, and manufacturing process suitable for mass production are required.
  • Another object of the present invention is to provide an efficient method for preparing the novel polypeptide intermediate and resin complex compound.
  • Another object of the present invention is to provide an efficient method for preparing a bioactive polypeptide using the novel polypeptide intermediate.
  • one embodiment of the present invention provides a novel polypeptide intermediate of the formula (1).
  • R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 Cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkylC 6-12 aryl, C 1-6 alkylC 6-12 aryloxycarbonyl and heteroaryl Selected from the group consisting of;
  • X is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
  • the aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
  • one embodiment of the present invention provides a novel resin complex compound of Formula 3:
  • a to D are protecting groups
  • a to D are each independently triphenylmethyl (Trt), tertiary butyl (tBu), t-butyloxycarbonyl (Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5- Selected from the group consisting of sulfonyl (Pbf),
  • R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
  • the aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
  • a to D are protecting groups
  • a to D are each independently triphenylmethyl (Trt), tertiary butyl (tBu), t-butyloxycarbonyl (Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5- Selected from the group consisting of sulfonyl (Pbf),
  • R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
  • the aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
  • the step of preparing a deprotected resin by deprotecting the protecting group using a piperidine solution in the cyclized peptide compound prepared by the above method in a polar aprotic solvent (2) adding protected amino acid, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide in a polar aprotic solvent to activate the protected amino acid; (3) adding and coupling an activated protected amino acid solution to the deprotected resin in the reactor; (4) repeating steps (1)-(3) until the peptide is formed; (5) cleaving the desired peptide from the resin while simultaneously deprotecting the protected resin using the cleavage cocktail; And (6) filtering the cleavage mixture from the resin, to provide a method for producing a bioactive polypeptide and a pharmaceutically acceptable salt.
  • novel polypeptide intermediate and the manufacturing process thereof according to the present invention can provide a novel polypeptide intermediate that can be utilized in bioactive polypeptide pharmaceuticals, and can be reproducible production of high-quality products suitable for mass production There is an advantage.
  • the protecting groups of amino acids used in the present invention are stable under peptide condensation conditions, are easily removable and do not affect peptide chains and substituents in the elimination reaction, as well as the racemization of any chiral centers present in the peptide. Anything that can be used can be used.
  • suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), 2- (4-nitrophenyl-sulfonyl) ethoxycarbonyl (NSC), t-butoxycarbonyl (Boc), benzyloxycarbono Neyl (Cbz), biphenylisopropyl-oxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, ( ⁇ , ⁇ ) -dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitro Phenylsulphenyl, 2-cyano-t-butyloxycarbonyl, and the like, but are not limited to these and other suitable protecting groups known in the art for this purpose may also be used within the scope of the present invention.
  • fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc) groups can be used.
  • a solid-phase peptide synthesis method using 9-fluoroenylmethoxycarbonyl (Fmoc) as an amino acid protecting group may be used.
  • Resin used in the reaction of all the steps of the present invention is a polymer support treated with a suitable linker, polystyrene (PS) resin or polystyrene-polyethylene glycol copolymer (PS-PEG copolymer) resin is preferred, but without limitation, other suitable resins known in the art for this purpose may also be used within the scope of the present invention.
  • PS polystyrene
  • PS-PEG copolymer polystyrene-polyethylene glycol copolymer
  • the resins usable in the present invention are, for example, aminomethyl resin, aminoethyl resin, aminobutyl resin, linkamide aminomethyl resin, linkamide aminoethyl resin, linkamide aminobutyl resin, linkamide MBHA resin, linkamide for polystyrene series.
  • Polar aprotic solvents used in the reactions of all stages of the present invention include, for example, dimethylformamide, dimethylacetamide, and the like, but are not limited to these other suitable polar aprotic solvents known in the art for this purpose. It is also possible to use within the scope of the present invention.
  • the polar aprotic solvent used in the reaction of all stages of the present invention may preferably be selected from the group consisting of dimethylformamide, dimethylacetamide and mixtures thereof.
  • step (1) the resin is swollen in a polar aprotic solvent.
  • step (2) deprotected resin is prepared by deprotecting the protecting group using a piperidine solution in a polar aprotic solvent.
  • the deprotected resin can be washed with a polar solvent.
  • the polar solvent used herein may be selected from the group consisting of dimethylformamide, dimethylacetamide, methanol, ethanol and mixtures thereof.
  • protected amino acids, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide are added in a polar aprotic solvent to activate the protected amino acids.
  • step (4) an activated protected amino acid solution is added to the deprotected resin in the reactor to perform a coupling reaction.
  • the coupled resin can be washed using a polar solvent.
  • the polar solvent used herein may be selected from the group consisting of dimethylformamide, dimethylacetamide, methanol, ethanol and mixtures thereof.
  • steps (2)-(6) are repeated until a peptide is formed.
  • step (5) may be carried out repeatedly 2 to 100 times until a desired length of peptide is formed, preferably 10 to 50 times, most preferably 14 to 30 times Can be.
  • step (6) the synthesized peptide is reacted with tetrakispalladium, N-methylaniline and penicsilane in a solvent to prepare a partially deprotected resin.
  • the solvent used in the reaction of step (6) may be selected from the group consisting of dichloromethane, chloroform and mixtures thereof.
  • a polypeptide intermediate is prepared by adding a coupling reagent and a coupling reagent in a polar aprotic solvent to perform a cyclization reaction.
  • the coupling reagent used in the reaction of step (7) is 1-hydroxy-1H-benzotriazole / 1,3-diisopropylcarbodiimide or HATU (1- [bis (dimethylamino) methylene] -1H -1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium) / N, N-diisopropylethyl
  • Other coupling reagents known in the art for this purpose may also be used within the scope of the present invention, although it may be selected from amines.
  • step (1) the deprotected resin is prepared by deprotecting the protecting group using the piperidine solution of the cyclized peptide compound prepared by the above method in a polar aprotic solvent.
  • step (2) protected amino acids, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide are added in a polar aprotic solvent to activate the protected amino acids.
  • step (3) an activated protected amino acid solution is added to the deprotected resin in the reactor to carry out the coupling reaction.
  • step (4) steps (1)-(3) are repeated until a peptide is formed.
  • step (4) may be performed repeatedly 1 to 50 times, preferably 1 to 30 times, until a peptide of a desired length is formed.
  • step (5) above the cleavage cocktail is used to deprotect the protected resin while simultaneously cleaving the desired peptide from the resin.
  • the cleavage cocktail of step (5) may comprise a solution of trifluoroacetic acid (TFA), one or more scavengers and dichloromethane.
  • TFA trifluoroacetic acid
  • scavengers one or more scavengers and dichloromethane.
  • the scavenger of step (5) is said scavenger is triisopropylsilane (TIPS), triethylsilane (TES), phenol, anisole, thioanisole, water, ethanedithiol (EDT), 1-dodecane Thiol, dithiothreitol (DTT) and indole may be selected from the group consisting of, but not limited to, other suitable scavengers known in the art for this purpose are also available within the scope of the present invention.
  • TIPS triisopropylsilane
  • TES triethylsilane
  • phenol anisole
  • thioanisole water
  • EDT ethanedithiol
  • DTT 1-dodecane Thiol
  • DTT dithiothreitol
  • indole may be selected from the group consisting of, but not limited to, other suitable scavengers known in the art for this purpose are also available within the scope
  • step (6) there is provided a method of preparing a bioactive polypeptide and a pharmaceutically acceptable salt comprising filtering the cleavage mixture from the resin.
  • novel polypeptide intermediate provided by the present invention and a pharmaceutical intermediate of high purity can be provided through the preparation method thereof, and the bioactive polypeptide prepared by using the same can also be used for the manufacture of high-quality pharmaceutical products with high purity and easy purification. This is possible.
  • a physiologically active polypeptide can be prepared by linear synthesis of the polypeptide followed by a cyclization reaction.
  • linearly synthesize 16 to 30 amino acids sequentially and perform a cyclization reaction of each linear synthesized polypeptide (16mer to 30mer).
  • the purity tends to decrease rapidly as the length of the linearly synthesized polypeptide increases.
  • the purification process becomes very difficult and thus yields tend to decrease rapidly.
  • the novel polypeptide intermediate and the physiologically active polypeptide prepared through the preparation method according to the present invention undergo a step of preparing a linear polypeptide up to 15mer, performing a cyclization reaction, and further synthesizing the remaining amino acids. Therefore, it has the advantage of synthesizing a high purity product compared to the existing manufacturing method.
  • the manufacturing method of the present invention not only facilitates the purification process after synthesis, but also has the advantage that the final manufacturing yield is greatly improved and is an efficient process suitable for commercial production.
  • amino acids referred to herein as abbreviations are described according to the IUPAC-IUB nomenclature.
  • Methionine-Met M; Phenylalanine-Phe, F; Proline-Pro, P;
  • Solid phase peptide synthesis methods including deprotection of amino acids, methods of cleaving peptides from resins, and SPPS methods, including purification thereof), as well as methods of detecting and characterizing the resulting peptides (LCMS, MALDI, and UPLC) Method).
  • N-terminal amino acids have an alpha amino group protected with Boc (eg Boc-His (Boc) -OH, or Boc-His (Trt) -OH for peptides having His at the N-terminus).
  • Boc eg Boc-His (Boc) -OH
  • Boc-His (Trt) -OH for peptides having His at the N-terminus.
  • link amide MBHA resin 80.0 g (0.31 mmol / g) of link amide MBHA resin and 480 ml of dimethylformamide were added to the vessel, stirred for 15 minutes, and filtered to remove dimethylformamide. This process was carried out twice. Through the above procedure, link amide MBHA resin was prepared.
  • Cys (Trt) -Fmoc activated in step (2) was added to the Fmoc deprotected link amide MBHA resin prepared in step (1), stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Resin-Cys (Trt) -Fmoc, was obtained.
  • Step (2) Activation of Thr (tBu) -Fmoc (T)
  • Thr (tBu) -Fmoc activated in step (2) was added to the Fmoc deprotected resin-Cys (Trt) prepared in step (1), followed by stirring at room temperature for 3 hours or more, followed by filtration.
  • 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration.
  • Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total.
  • 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total.
  • Step (2) Activation of Asn (Trt) -Fmoc (N)
  • Asn (Trt) -Fmoc activated in step (2) was added to the Fmoc deprotected resin-Cys (Trt) -Thr (tBu) prepared in step (1), followed by stirring at room temperature for 3 hours or more, followed by filtration. . 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Fmoc, was obtained.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc (Poly 4mer Synthesis)
  • step (2) After adding the activated Met-Fmoc in step (2) to the Fmoc deprotected resin-Cys (Trt) -Thr (tBu) -Asn (Trt) prepared in step (1) and stirred at room temperature for 3 hours or more. Filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc, was obtained.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc (Poly 5mer Synthesis)
  • Lemo-Fmoc activated in step (2) was added to the Fmoc deprotected resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met prepared in step (1), followed by stirring at room temperature for 3 hours or more. And then filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc was obtained.
  • Trp (Boc) -Fmoc 39.2 g of Trp (Boc) -Fmoc, 1H-benzotriazole, 16.7 g, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Trp (Boc) -Fmoc was activated.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc (Poly 6mer Synthesis)
  • step (2) The Tmo (Boc) -Fmoc activated in step (2) was added to the Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu prepared in step (1).
  • the mixture was stirred at least 3 hours and filtered.
  • 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration.
  • Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total.
  • 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total.
  • Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc was obtained.
  • Step (2) Activation of Gln (Trt) -Fmoc (Q)
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc (Poly 7mer Synthesis)
  • Val-Fmoc 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Val-Fmoc, the target compound, was activated.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Fmoc (Poly 8mer Synthesis)
  • step (1) The Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) prepared in step (1) was activated in step (2). Val-Fmoc was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Fmoc (Poly 9mer Synthesis)
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Fmoc (Poly 10mer compound)
  • Step (2) Activation of Lys (Boc) -Fmoc (K)
  • Glu (OAll) -Fmoc Into the vessel, 30.5 g of Glu (OAll) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added and stirred to dissolve completely. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Glu (OAll) -Fmoc was activated.
  • Step (2) -1 Preparation of Cyclic Polypeptide (cyclic Poly 15mer Synthesis)
  • Step (2) -2 Preparation of Cyclic Polypeptide (cyclic Poly 15mer Synthesis)
  • HATU 1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzo in a container 56.6 g of triazole tetramethyl uronium,) and 480 ml of dimethylformamide were added and stirred to dissolve completely. 51.8 mL of N, N-diisopropylethylamine was added to the dissolved reaction solution, and the mixture was stirred at room temperature for 5 minutes.
  • reaction solution prepared in the polypeptide (partial deprotection 15mer) prepared in step (1) was added thereto, stirred at room temperature for 3 hours, and filtered.
  • 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration.
  • Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total.
  • 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total.
  • 480 ml of 20% piperidine was added to the cyclized polypeptide (cyclic poly 15mer synthesis) prepared in Example 3, stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total.
  • Step (2) Activation of Asp (OtBu) -Fmoc (D)
  • Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total.
  • Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total.
  • Step (2) Activation of Lys (Boc) -Fmoc (K)
  • Step (2) Activation of Ser (tBu) -Fmoc (S)
  • Step (2) Activation of Asp (OtBu) -Fmoc (D)
  • Step (2) Activation of Ser (tBu) -Fmoc (S)
  • Step (2) Activation of Thr (tBu) -Fmoc (T)
  • Step (2) Activation of Thr (tBu) -Fmoc (T)
  • Step (2) Activation of Gln (Trt) -Fmoc (Q)
  • Step (2) Activation of Aib-Fmoc
  • Example 5 NH 2 CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu] -Asp-Leu-Tyr-Lys-Ser- Preparation of Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-NH2, TFA (protector and resin cleavage, cleavage)
  • vessel 2 165 ml of trifluoroacetic acid, 10 ml of phenol, 10 ml of distilled water, 10 ml of thioanisole, and 5 ml of 1,2-ethanedithiol were added to the vessel 2 and stirred for 10 minutes. 20 g of the dried cyclocyclized polypeptide 30mer was added to the vessel 1, and the prepared reaction solution was added to the vessel 1, followed by stirring at room temperature for 1 hour 30 minutes. 3.0 L of methyl tertiary butyl ether was put into the container 3, and it cooled to 0-1 degreeC under nitrogen atmosphere.
  • the reaction liquid temperature of the vessel 1 was cooled to 5 ° C., 2.0 L of the cooled reactant methyl tertiary butyl ether of the vessel 3 was added thereto, and stirred for 10 minutes.
  • the reaction solution is filtered and washed twice with 400 ml of cooled methyl tertiary butyl ether. Crystals of the filter were dried under nitrogen atmosphere for 10 minutes. Dried crystals were added to the vessel 1, 400 ml of distilled water was added thereto, followed by stirring at room temperature for 10 minutes.
  • the reaction was filtered and washed with distilled water 600ml to give the target compound NH 2 CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu] -Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-NH 2 , TFA was obtained.
  • Rink amide MBHA resin 80.0g (0.31 mmol / g) and 480 ml of dimethylformamide were added to the vessel, stirred for 15 minutes, and filtered to remove dimethylformamide. This process was carried out twice.
  • Rink amide MBHA resin was prepared by the above procedure.
  • Cys (Trt) -Fmoc activated in step (2) was added to the Fmoc deprotected Rink amide MBHA resin prepared in step (1), stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound Resin-Cys (Trt) -Fmoc was obtained.
  • Step (2) Activation of Thr (tBu) -Fmoc (T)
  • Step (2) Activation of Asn (Trt) -Fmoc (N)
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 3). Trt was obtained.
  • Met-Fmoc 18.4g was carried out in the same manner as in step (2) of Synthesis 1) to activate Met-Fmoc as a target compound.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc (Poly 4mer Synthesis)
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 4). Trt) -Met was obtained.
  • Leu-Fmoc 17.5g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Leu-Fmoc.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc (Poly 5mer Synthesis)
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 5). Trt) -Met-Leu was obtained.
  • Trp (Boc) -Fmoc 26.1g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Trp (Boc) -Fmoc.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc (Poly 6mer Synthesis)
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (target compound) in the same manner as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 6). Trt) -Met-Leu-Trp (Boc) was obtained.
  • Step (2) Activation of Gln (Trt) -Fmoc (Q)
  • Gln (Trt) -Fmoc 30.3g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Gln (Trt) -Fmoc.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc (Poly 7mer Synthesis)
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (target compound) in the same manner as step (1) of Synthesis 1) with the polypeptide (poly 7mer synthesis) prepared in Synthesis 7). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) was obtained.
  • Val-Fmoc 16.8g into the container was carried out in the same manner as in step (2) of synthesis 1) to activate the target compound Val-Fmoc.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Fmoc (Poly 8mer Synthesis)
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 8). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val was obtained.
  • Phe-Fmoc 19.2g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Phe-Fmoc.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Fmoc (Poly 9mer Synthesis)
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 9). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe was obtained.
  • Step (3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Fmoc (Poly 10mer compound)
  • Lys (Alloc) -Fmoc 22.4g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Lys (Alloc) -Fmoc.
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 11). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) was obtained.
  • Ala-Fmoc 15.4g was activated in the same manner as in step (2) of Synthesis 1) to activate Ala-Fmoc, the target compound.
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 12). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala was obtained.
  • Arg (Pbf) -Fmoc 32.2g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Arg (Pbf) -Fmoc.
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 13). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) was obtained.
  • Step (2) Activation of Lys (Boc) -Fmoc (K)
  • Lys (Boc) -Fmoc 23.2g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Lys (Boc) -Fmoc.
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 14). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) was obtained.
  • Glu (OAll) -Fmoc 20.3g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Glu (OAll) -Fmoc.
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 15). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll).
  • Step (2) Activation of Asp (OtBu) -Fmoc (D)
  • Asp (OtBu) -Fmoc 20.4g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Asp (OtBu) -Fmoc as a target compound.
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 16). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) was obtained.
  • Leu-Fmoc 17.5g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Leu-Fmoc.
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 17).
  • Trt -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu was obtained.
  • Tyr (tBu) -Fmoc 22.8g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Tyr (tBu) -Fmoc.
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 18).
  • Trt -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) was obtained.
  • Step (2) Activation of Lys (Boc) -Fmoc (K)
  • Lys (Boc) -Fmoc 23.2g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Lys (Boc) -Fmoc.
  • Trt -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) was obtained.
  • Step (2) Activation of Ser (tBu) -Fmoc (S)
  • Ser (tBu) -Fmoc 19.0g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Ser (tBu) -Fmoc as a target compound.
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (target compound) in the same manner as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 20).
  • Trt -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) was obtained.
  • Tyr (tBu) -Fmoc 22.8g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Tyr (tBu) -Fmoc.
  • Trt Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 21).
  • Trt -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) was obtained.
  • Step (2) Activation of Asp (OtBu) -Fmoc (D)
  • Asp (OtBu) -Fmoc 20.4g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Asp (OtBu) -Fmoc as a target compound.
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 22).
  • Trt -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) was obtained.
  • Step (2) Activation of Ser (tBu) -Fmoc (S)
  • Ser (tBu) -Fmoc 19.0g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Ser (tBu) -Fmoc as a target compound.
  • Step (2) Activation of Thr (tBu) -Fmoc (T)
  • Phe-Fmoc 19.2g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Phe-Fmoc.
  • Step (2) Activation of Thr (tBu) -Fmoc (T)
  • Gly-Fmoc 14.7g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Gly-Fmoc.
  • Trt Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () with the polypeptide prepared in Synthesis 27) in the same manner as in step (1) of Synthesis 1). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly .
  • Step (2) Activation of Gln (Trt) -Fmoc (Q)
  • Gln (Trt) -Fmoc 30.3g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Gln (Trt) -Fmoc.
  • Step (2) Activation of Aib-Fmoc
  • Aib-Fmoc 16.1g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Aib-Fmoc as a target compound.
  • His (Trt) -Boc 30.7g was synthesized in the same manner as in step (2) of Synthesis 1) to activate His (Trt) -Boc as a target compound.

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Abstract

The present invention relates to a novel intermediate used for a physiologically active polypeptide and a method for preparing same. The novel intermediate may be effectively used as an intermediate for the preparation of physiologically active polypeptide pharmaceuticals, and may be efficiently used for the preparation of high quality pharmaceuticals by providing a polypeptide intermediate of high yield and high purity.

Description

생리활성 폴리펩타이드에 사용되는 신규한 중간체 및 이의 제조방법Novel intermediates used in bioactive polypeptides and methods for their preparation
본 발명은 생리활성 폴리펩타이드에 사용되는 신규한 중간체 및 이의 제조방법에 관한 것이다. 보다 구체적으로 본 발명은 생리활성 폴리펩타이드를 보다 안전하고 효율적으로 제조하는 방법 및 이에 사용되는 신규한 폴리펩타이드 중간체 및 이의 제조방법에 관한 것이다.The present invention relates to novel intermediates used in physiologically active polypeptides and methods for their preparation. More specifically, the present invention relates to a method for producing a bioactive polypeptide more safely and efficiently, and to a novel polypeptide intermediate and a method for producing the same.
비만과 2형 당뇨를 포함하는 당뇨관련 질병은 현대사회에서 발생하는 대표적인 대사질환 중 하나로서 전세계적으로 건강에 대한 중요한 위협요소로 인식되고 있으며, 이에 따른 경제적 비용도 급증하는 추세다.Diabetes-related diseases, including obesity and type 2 diabetes, are one of the major metabolic diseases occurring in modern society and are recognized as an important threat to health around the world, and economic costs are increasing rapidly.
이러한 비만과 당뇨의 치료에 사용할 수 있는 의약품의 개발 연구가 진행되어 왔으나, 이들은 치명적인 부작용을 나타내거나 비만치료효과가 미비하다는 단점을 가지고 있다. 따라서, 종래 치료제의 문제점을 해소할 수 있는 연구가 활발히 진행되고 있으며, 최근에는 글루카곤 유도체에 관심이 집중되고 있다. 글루카곤은 약물 치료 또는 질병, 호르몬이나 효소 결핍 등의 원인으로 혈당이 떨어지기 시작하면 췌장에서 생산된다. 글루카곤은 간에서 글리코겐을 분해하여 글루코스를 방출하도록 신호하고, 혈당 수준을 정상 수준까지 높이는 역할을 한다. 뿐만 아니라, 글루카곤은 혈당상승효과 이외에 식욕억제 및 지방세포의 호르몬 민감성 리파제(hormone-sensitive lipase)를 활성화시켜 지방분해를 촉진하여 항비만 효과를 나타냄이 보고되었고, 이와 관련한 다양한 연구가 진행 중이다.Research into the development of medicines that can be used for the treatment of obesity and diabetes has been conducted, but these have the disadvantages of fatal side effects or ineffective obesity treatment. Therefore, studies are being actively conducted to solve the problems of the conventional therapeutics, and recently, attention has been focused on glucagon derivatives. Glucagon is produced in the pancreas when blood sugar begins to drop due to medication or disease, hormone or enzyme deficiency. Glucagon signals the breakdown of glycogen in the liver to release glucose and raises blood sugar levels to normal levels. In addition, glucagon has been reported to exhibit anti-obesity effects by promoting lipolysis by activating hormone-sensitive lipase of appetite suppression and adipocytes in addition to the effect of increasing blood glucose. Various studies are in progress.
일례로, 대한민국 공개특허 제10-2017-0080521호에서는 글루카곤, GLP-1 및 GIP 수용체 모두에 활성을 갖는 삼중 활성체 및 이의 용도에 대하여 개시하고 있다. 이러한 펩타이드는 천연형 글루카곤 서열에서 적어도 하나 이상의 아미노산에 치환(substitution), 추가(addition), 제거(deletion), 수식 (modification) 및 이들의 조합으로부터 이루어질 수 있고, 보다 구체적으로, 하기 일반식 1로 표시된 아미노산 서열을 포함하는, 분리된 펩타이드를 개시하고 있다. Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (일반식 1, 서열번호 103)For example, Korean Patent Laid-Open Publication No. 10-2017-0080521 discloses a triple activator having activity on both glucagon, GLP-1, and GIP receptor and use thereof. Such peptides may be formed from substitution, addition, removal, modification, and combinations thereof of at least one or more amino acids in a native glucagon sequence, and more specifically, in Formula 1 An isolated peptide is disclosed comprising the indicated amino acid sequence. Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp- Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (Formula 1, SEQ ID NO: 103)
상기의 펩타이드는 그 길이에 따라 이 분야에서 알려진 방법, 예를 들어 자동 펩타이드 합성기에 의한 합성, 유전자 조작기술, 임의의 다른 방법에 의하여 제조할 수 있다. 이러한 다양한 제조방법으로 제조한 펩타이드를 의약품으로 사용하기 위해서는 고순도의 품질과, 상업화에 적합한 수율 및 대량생산에 적합한 제조공정이 필요하다. The peptides can be prepared according to their length by methods known in the art, such as by synthetic peptide synthesizers, by genetic engineering techniques, or by any other method. In order to use the peptides prepared by such various manufacturing methods as pharmaceuticals, high purity quality, yield suitable for commercialization, and manufacturing process suitable for mass production are required.
따라서, 이러한 생리활성 폴리펩타이드의 효율적인 제조를 가능하게 하는 신규한 폴리펩타이드 중간체 및 이를 제공할 수 있는 효율적인 제조방법이 요구된다. 이에 본 발명자들은 생리활성 폴리펩타이드를 보다 안전하고 효율적으로 제조하는 방법에 대해 연구한 결과, 신규한 폴리펩타이드 중간체 및 이의 제조방법을 개발하여 본 발명을 완성하였다.Therefore, there is a need for novel polypeptide intermediates that allow for the efficient production of such bioactive polypeptides and efficient methods for providing them. Accordingly, the present inventors have studied the method for producing a bioactive polypeptide more safely and efficiently, and as a result, a novel polypeptide intermediate and a method for preparing the same have been completed.
[선행기술문헌][Preceding technical literature]
[특허문헌][Patent Documents]
대한민국 공개특허 제10-2017-0080521호 (2017.07.10), 글루카곤, GLP-1 및 GIP 수용체 모두에 활성을 갖는 삼중 활성체Republic of Korea Patent Publication No. 10-2017-0080521 (2017.07.10), triple activator having activity on both glucagon, GLP-1 and GIP receptor
본 발명의 목적은 생리활성 폴리펩타이드에 사용되는 신규한 폴리펩타이드 중간체 및 레진 복합체 화합물을 제공하는 것이다. It is an object of the present invention to provide novel polypeptide intermediates and resin complex compounds for use in bioactive polypeptides.
본 발명의 다른 목적은 상기 신규한 폴리펩타이드 중간체 및 레진 복합체 화합물의 효율적인 제조 방법을 제공하는 것이다.Another object of the present invention is to provide an efficient method for preparing the novel polypeptide intermediate and resin complex compound.
본 발명의 또 다른 목적은 상기 신규한 폴리펩타이드 중간체를 사용하는 생리활성 폴리펩타이드의 효율적인 제조 방법을 제공하는 것이다.Another object of the present invention is to provide an efficient method for preparing a bioactive polypeptide using the novel polypeptide intermediate.
상기 목적을 달성하기 위하여, 본 발명의 일 구체예에서는 하기 화학식 1의 신규한 폴리펩타이드 중간체를 제공한다. In order to achieve the above object, one embodiment of the present invention provides a novel polypeptide intermediate of the formula (1).
[화학식 1][Formula 1]
Figure PCTKR2019008935-appb-img-000001
Figure PCTKR2019008935-appb-img-000001
: R-Cyclo(-Glu-Lys-Arg-Ala-Lys)-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-X.: R-Cyclo (-Glu-Lys-Arg-Ala-Lys) -Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-X.
여기서, R은 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되고;Wherein R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 Cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkylC 6-12 aryl, C 1-6 alkylC 6-12 aryloxycarbonyl and heteroaryl Selected from the group consisting of;
X는 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되며;X is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
상기 언급된 치환체들은 추가적으로 H, 할로젠, 시아노, 직쇄형 또는 분지형의 C 1-6알킬, 직쇄형 또는 분지형의 C 2-10알켄일, C 3-10사이클로알킬, 할로C 1-5알킬, 하이드록시C 1-6알킬, 아미노, 모노 또는 디C 1-6알킬아미노, 옥소, 하이드록시, C 1-6알콕시, C 6-12아릴설포닐 및 C 1-6알킬설포닐로 이루어진 군에서 선택된 1종 이상의 동일하거나 상이한 치환기로 치환될 수 있다.The aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
또한, 본 발명의 일 구체예에서는 화학식 3의 신규한 레진 복합체 화합물을 제공한다:In addition, one embodiment of the present invention provides a novel resin complex compound of Formula 3:
[화학식 3][Formula 3]
Figure PCTKR2019008935-appb-img-000002
Figure PCTKR2019008935-appb-img-000002
여기서, A 내지 D는 보호기이며;Wherein A to D are protecting groups;
A 내지 D는 각각 독립적으로 트리페닐메틸(Trt), 터셔리부틸(tBu), t-부틸옥시카보닐(Boc) 및 2,2,4,6,7-펜타메틸디하이드로벤조퓨란-5-설포닐(Pbf)로 이루어진 군으로부터 선택되고,A to D are each independently triphenylmethyl (Trt), tertiary butyl (tBu), t-butyloxycarbonyl (Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5- Selected from the group consisting of sulfonyl (Pbf),
R은 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되고;R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
X'은 레진이며;X 'is a resin;
상기 언급된 치환체들은 추가적으로 H, 할로젠, 시아노, 직쇄형 또는 분지형의 C 1-6알킬, 직쇄형 또는 분지형의 C 2-10알켄일, C 3-10사이클로알킬, 할로C 1-5알킬, 하이드록시C 1-6알킬, 아미노, 모노 또는 디C 1-6알킬아미노, 옥소, 하이드록시, C 1-6알콕시, C 6-12아릴설포닐 및 C 1-6알킬설포닐로 이루어진 군에서 선택된 1종 이상의 동일하거나 상이한 치환기로 치환될 수 있다.The aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
또한, 본 발명의 일 구체예에서는 (1) 극성 비양성자성 용매 중에서 레진을 팽윤시키는 단계; (2) 극성 비양성자성 용매 중에서 피페리딘 용액을 사용하여 보호기를 탈보호화시켜 탈보호화된 레진을 제조하는 단계; (3) 극성 비양성자성 용매 중에 보호화된 아미노산, 1-히드록시-1H-벤조트리아졸 및 1,3-디이소프로필카보디이미드를 가하여 보호화된 아미노산을 활성화시키는 단계; (4) 반응기 내 탈보호화된 레진에 활성화된 보호화된 아미노산 용액을 가하여 커플링하는 단계; (5) 펩타이드가 형성될 때까지 단계 (2)-(4)를 반복하는 단계; (6) 합성된 펩타이드를 용매 하에서 테트라키스팔라듐, N-메틸아닐린 및 페니실란과 반응시켜 부분 탈보호화된 레진을 제조하는 단계; 및 (7) 극성 비양성자성 용매 중에 합성된 펩타이드와 커플링 시약을 가하여 고리화하는 단계를 포함하여 하기 화학식 3의 레진 복합체 화합물을 제조하는 방법을 제공한다.In addition, in one embodiment of the present invention (1) swelling the resin in a polar aprotic solvent; (2) deprotecting the protecting group using a piperidine solution in a polar aprotic solvent to prepare a deprotected resin; (3) adding protected amino acids, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide in a polar aprotic solvent to activate the protected amino acids; (4) adding and coupling an activated protected amino acid solution to the deprotected resin in the reactor; (5) repeating steps (2)-(4) until the peptide is formed; (6) reacting the synthesized peptide with tetrakispalladium, N-methylaniline and penicsilane in a solvent to produce partially deprotected resin; And (7) adding a peptide and a coupling reagent synthesized in a polar aprotic solvent to cyclize it.
[화학식 3][Formula 3]
Figure PCTKR2019008935-appb-img-000003
Figure PCTKR2019008935-appb-img-000003
여기서, A 내지 D는 보호기이며;Wherein A to D are protecting groups;
A 내지 D는 각각 독립적으로 트리페닐메틸(Trt), 터셔리부틸(tBu), t-부틸옥시카보닐(Boc) 및 2,2,4,6,7-펜타메틸디하이드로벤조퓨란-5-설포닐(Pbf)로 이루어진 군으로부터 선택되고,A to D are each independently triphenylmethyl (Trt), tertiary butyl (tBu), t-butyloxycarbonyl (Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5- Selected from the group consisting of sulfonyl (Pbf),
R은 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되고;R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
X'은 레진이며;X 'is a resin;
상기 언급된 치환체들은 추가적으로 H, 할로젠, 시아노, 직쇄형 또는 분지형의 C 1-6알킬, 직쇄형 또는 분지형의 C 2-10알켄일, C 3-10사이클로알킬, 할로C 1-5알킬, 하이드록시C 1-6알킬, 아미노, 모노 또는 디C 1-6알킬아미노, 옥소, 하이드록시, C 1-6알콕시, C 6-12아릴설포닐 및 C 1-6알킬설포닐로 이루어진 군에서 선택된 1종 이상의 동일하거나 상이한 치환기로 치환될 수 있다.The aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
또한, 본 발명의 일 구체예에서는 (1) 상기의 방법으로 제조한 고리화된 펩타이드 화합물을 극성 비양성자성 용매 중에서 피페리딘 용액을 사용하여 보호기를 탈보호화시켜 탈보호화된 레진을 제조하는 단계; (2) 극성 비양성자성 용매 중에 보호화된 아미노산, 1-히드록시-1H-벤조트리아졸 및 1,3-디이소프로필카보디이미드를 가하여 보호화된 아미노산을 활성화시키는 단계; (3) 반응기 내 탈보호화된 레진에 활성화된 보호화된 아미노산 용액을 가하여 커플링하는 단계; (4) 펩타이드가 형성될 때까지 단계 (1)-(3)을 반복하는 단계; (5) 절단 칵테일을 사용하여 보호화된 레진을 탈보호화시키는 동시에 레진으로부터 원하는 펩타이드를 절단하는 단계; 및 (6) 레진으로부터 절단 혼합물을 여과하는 단계를 포함하는, 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염을 제조하는 방법을 제공한다.In addition, in one embodiment of the present invention (1) the step of preparing a deprotected resin by deprotecting the protecting group using a piperidine solution in the cyclized peptide compound prepared by the above method in a polar aprotic solvent ; (2) adding protected amino acid, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide in a polar aprotic solvent to activate the protected amino acid; (3) adding and coupling an activated protected amino acid solution to the deprotected resin in the reactor; (4) repeating steps (1)-(3) until the peptide is formed; (5) cleaving the desired peptide from the resin while simultaneously deprotecting the protected resin using the cleavage cocktail; And (6) filtering the cleavage mixture from the resin, to provide a method for producing a bioactive polypeptide and a pharmaceutically acceptable salt.
상술한 방법으로부터 본 발명의 일 구체예에서는 하기 화학식 2의 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염을 제조하는 방법이 제공된다.In one embodiment of the present invention from the above-described method is provided a method for preparing a physiologically active polypeptide of the formula (2) and a pharmaceutically acceptable salt.
[화학식 2][Formula 2]
Figure PCTKR2019008935-appb-img-000004
Figure PCTKR2019008935-appb-img-000004
: H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Ser-Lys-Tyr-Leu-Asp- Cyclo(-Glu-Lys-Arg-Ala-Lys)-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-NH 2 : H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Ser-Lys-Tyr-Leu-Asp- Cyclo (-Glu-Lys-Arg-Ala- Lys) -Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-NH 2
본 발명에 따른 신규한 폴리펩타이드 중간체 및 이의 제조공정은 생리활성 폴리펩타이드 의약품에 활용할 수 있는 신규한 폴리펩타이드 중간체를 제공할 수 있고, 대량 생산에 적합하고 효율적이며 고품질의 제품을 재현성있게 생산할 수 있는 장점이 있다.The novel polypeptide intermediate and the manufacturing process thereof according to the present invention can provide a novel polypeptide intermediate that can be utilized in bioactive polypeptide pharmaceuticals, and can be reproducible production of high-quality products suitable for mass production There is an advantage.
본 발명의 신규한 폴리펩타이드 중간체의 제조방법을 단계별로 보다 구체적으로 설명하면 다음과 같다.Hereinafter, the method of preparing the novel polypeptide intermediate of the present invention will be described in more detail as follows.
본 발명에 사용되는 아미노산의 보호기는 펩타이드 축합반응 조건에서 안정성을 가지며, 용이하게 제거 가능하고 제거 반응시 펩타이드 사슬 및 치환체에 영향을 주지 않을 뿐 아니라 펩타이드에 존재하는 임의의 키랄 센터의 라세미체화를 일으키지 않는 것이라면 어느 것이라도 사용 가능하다. 예컨대, 적합한 보호기들로는 9-플루오레닐메틸옥시카보닐 (Fmoc), 2-(4-니트로페닐-술포닐)에톡시카르보닐 (NSC), t-부톡시카보닐 (Boc), 벤질옥시카보닐 (Cbz), 비페닐이소프로필-옥시카보닐, t-아밀옥시카보닐, 이소보르닐옥시카보닐, (α,α)-디메틸-3,5-디메톡시벤질옥시카보닐, O-니트로페닐설페닐, 2-시아노-t-부틸옥시카보닐 등일 수 있으나, 이들로 제한되는 것은 아니며 이러한 목적으로 당업계에 알려진 적합한 다른 보호기들 또한 본 발명의 범위 내에서 사용 가능하다. 바람직하게는 플루오레닐메틸옥시카보닐 (Fmoc) 또는 tert-부틸옥시카보닐 (Boc) 기를 사용할 수 있다.The protecting groups of amino acids used in the present invention are stable under peptide condensation conditions, are easily removable and do not affect peptide chains and substituents in the elimination reaction, as well as the racemization of any chiral centers present in the peptide. Anything that can be used can be used. For example, suitable protecting groups include 9-fluorenylmethyloxycarbonyl (Fmoc), 2- (4-nitrophenyl-sulfonyl) ethoxycarbonyl (NSC), t-butoxycarbonyl (Boc), benzyloxycarbono Neyl (Cbz), biphenylisopropyl-oxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (α, α) -dimethyl-3,5-dimethoxybenzyloxycarbonyl, O-nitro Phenylsulphenyl, 2-cyano-t-butyloxycarbonyl, and the like, but are not limited to these and other suitable protecting groups known in the art for this purpose may also be used within the scope of the present invention. Preferably fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc) groups can be used.
또한, 본 발명의 일 구체예에서는 아미노산의 보호기 중 9-플루오로에닐메톡시카보닐(Fmoc)을 아미노산 보호기로 사용하는 고체상 펩타이드 합성법을 이용할 수 있다.In addition, in one embodiment of the present invention, a solid-phase peptide synthesis method using 9-fluoroenylmethoxycarbonyl (Fmoc) as an amino acid protecting group may be used.
본 발명의 모든 단계의 반응에 사용되는 레진은 적절한 링커로 처리되어 있는 고분자 지지체로서, 폴리스티렌 (PS) 계열 레진 또는 폴리스티렌-폴리에틸렌 글리콜 공중합체 (PS-PEG copolymer) 계열의 레진이 바람직하지만, 이들로 제한되는 것은 아니며, 이러한 목적으로 당업계에 알려진 적합한 다른 레진들 또한, 본 발명의 범위 내에서 사용 가능하다. Resin used in the reaction of all the steps of the present invention is a polymer support treated with a suitable linker, polystyrene (PS) resin or polystyrene-polyethylene glycol copolymer (PS-PEG copolymer) resin is preferred, but Without limitation, other suitable resins known in the art for this purpose may also be used within the scope of the present invention.
본 발명에 사용 가능한 레진은 예컨대, 폴리스티렌 계열의 경우 아미노메틸 레진, 아미노에틸 레진, 아미노부틸 레진, 링크 아미드 아미노메틸 레진, 링크 아미드 아미노에틸 레진, 링크 아미드 아미노부틸 레진, 링크 아미드 MBHA 레진, 링크 아미드 레진, 2-클로로트리틸-N-Fmoc-하드록실아민 레진, HMPA-AM 레진, HMPB 레진, 2-클로로트리틸 레진, 4-카복시트리틸 레진, Wang 레진, PAL 레진, 4-(하이드록시메틸)페녹시아세트산 레진, Sieber 아미드 레진 이고, 폴리스티렌-폴리에틸렌 글리콜 공중합체 계열의 경우, TentaGel S 레진, TentaGel R 레진, TentaGel XV 레진, TentaGel MB 레진, TentaGel HL 레진, TentaGel B 레진, TentaGel M 레진 , TentaGel N 레진, TentaGel PAP 레진, 링크 아미드 TentaGel S 레진 (TentaGel S RAM), 링크 아미드 TentaGel R 레진 (TentaGel R RAM), 링크 아미드 TentaGel XV 레진 (TentaGel XV - RAM), 링크 아미드 TentaGel MB 레진 (TentaGel MB - RAM), 링크 아미드 TentaGel HL 레진 (TentaGel HL RAM), 링크 아미드 TentaGel B 레진 (TentaGel B RAM), 링크 아미드 TentaGel M 레진 (TentaGel M RAM), 링크 아미드 TentaGel N 레진 (TentaGel N RAM), 링크 아미드 TentaGel PAP 레진(TentaGel PAP RAM) 및 다양하게 치환된 HypoGel 200과 400 레진이다.The resins usable in the present invention are, for example, aminomethyl resin, aminoethyl resin, aminobutyl resin, linkamide aminomethyl resin, linkamide aminoethyl resin, linkamide aminobutyl resin, linkamide MBHA resin, linkamide for polystyrene series. Resin, 2-Chlorotrityl-N-Fmoc-hydroxylamine Resin, HMPA-AM Resin, HMPB Resin, 2-Chlorotrityl Resin, 4-Carboxytrityl Resin, Wang Resin, PAL Resin, 4- (Hydroxy Methyl) phenoxyacetic acid resin, Sieber amide resin, for polystyrene-polyethylene glycol copolymer series, TentaGel S resin, TentaGel R resin, TentaGel XV resin, TentaGel MB resin, TentaGel HL resin, TentaGel B resin, TentaGel M resin, TentaGel N resin, TentaGel PAP resin, Link amide TentaGel S resin (TentaGel S RAM), Link amide TentaGel R resin (TentaGel R RAM), Link amide TentaGel XV resin (TentaGel XV-RAM) , Link amide TentaGel MB resin (TentaGel MB-RAM), link amide TentaGel HL resin (TentaGel HL RAM), link amide TentaGel B resin (TentaGel B RAM), link amide TentaGel M resin (TentaGel M RAM), link amide TentaGel N RAM Resins (TentaGel N RAM), link amide TentaGel PAP resins and various substituted HypoGel 200 and 400 resins.
본 발명의 모든 단계의 반응에 사용되는 극성 비양성자성 용매는 예컨대, 디메틸포름아미드, 디메틸아세트아미드 등이 있으나, 이들로 제한되는 것은 아니며 이러한 목적으로 당업계에 알려진 적합한 다른 극성 비양성자성 용매들 또한 본 발명의 범위 내에서 사용 가능하다. Polar aprotic solvents used in the reactions of all stages of the present invention include, for example, dimethylformamide, dimethylacetamide, and the like, but are not limited to these other suitable polar aprotic solvents known in the art for this purpose. It is also possible to use within the scope of the present invention.
본 발명의 일 구체예에 따르면, 본 발명의 모든 단계의 반응에 사용되는 극성 비양성자성 용매는 바람직하게는 디메틸포름아미드, 디메틸아세트아미드 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다.According to one embodiment of the present invention, the polar aprotic solvent used in the reaction of all stages of the present invention may preferably be selected from the group consisting of dimethylformamide, dimethylacetamide and mixtures thereof.
본 발명의 상기 화학식 1의 펩타이드 중간체 화합물을 제조하는 방법을 단계별로 보다 구체적으로 설명하면 다음과 같다.Hereinafter, the method of preparing the peptide intermediate compound of Chemical Formula 1 of the present invention will be described in more detail as follows.
상기 단계 (1)에서는, 극성 비양성자성 용매 중에서 레진을 팽윤시킨다.In step (1), the resin is swollen in a polar aprotic solvent.
상기 단계 (2)에서는, 극성 비양성자성 용매 중에서 피페리딘 용액을 사용하여 보호기를 탈보호화시켜 탈보호화된 레진을 제조한다.In step (2), deprotected resin is prepared by deprotecting the protecting group using a piperidine solution in a polar aprotic solvent.
상기 단계 (2)의 반응 후, 극성 용매를 사용하여 탈보호화된 레진을 세척할 수 있다. 여기에 사용하는 극성 용매는 디메틸포름아미드, 디메틸아세트아미드, 메탄올, 에탄올 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다. 상기 단계 (3)에서는, 극성 비양성자성 용매 중에 보호화된 아미노산, 1-히드록시-1H-벤조트리아졸 및 1,3-디이소프로필카보디이미드를 가하여 보호화된 아미노산을 활성화시킨다.After the reaction of step (2), the deprotected resin can be washed with a polar solvent. The polar solvent used herein may be selected from the group consisting of dimethylformamide, dimethylacetamide, methanol, ethanol and mixtures thereof. In step (3), protected amino acids, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide are added in a polar aprotic solvent to activate the protected amino acids.
상기 단계 (4)에서는, 반응기 내 탈보호화된 레진에 활성화된 보호화된 아미노산 용액을 가하여 커플링 반응을 수행한다.In step (4), an activated protected amino acid solution is added to the deprotected resin in the reactor to perform a coupling reaction.
상기 단계 (4)의 반응 후, 극성 용매를 사용하여 커플링한 레진을 세척할 수 있다. 여기에 사용하는 극성 용매는 디메틸포름아미드, 디메틸아세트아미드, 메탄올, 에탄올 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다.After the reaction of step (4), the coupled resin can be washed using a polar solvent. The polar solvent used herein may be selected from the group consisting of dimethylformamide, dimethylacetamide, methanol, ethanol and mixtures thereof.
상기 단계 (5)에서는, 펩타이드가 형성될 때까지 단계 (2)-(6)을 반복하여 수행한다. In step (5), steps (2)-(6) are repeated until a peptide is formed.
상기 단계 (5)의 반응은, 원하는 길이의 펩타이드가 형성될 때까지 2 내지 100번 반복하여 수행할 수 있고, 바람직하게는 10 내지 50번, 가장 바람직하게는 14 내지 30번을 반복하여 수행할 수 있다.The reaction of step (5) may be carried out repeatedly 2 to 100 times until a desired length of peptide is formed, preferably 10 to 50 times, most preferably 14 to 30 times Can be.
상기 단계 (6)에서는, 합성된 펩타이드를 용매 하에서 테트라키스팔라듐, N-메틸아닐린 및 페니실란과 반응시켜 부분 탈보호된 레진을 제조한다.In step (6), the synthesized peptide is reacted with tetrakispalladium, N-methylaniline and penicsilane in a solvent to prepare a partially deprotected resin.
상기 단계 (6)의 반응에 사용되는 용매는 디클로로메탄, 클로로포름 및 이들의 혼합물로 이루어진 군으로부터 선택될 수 있다. The solvent used in the reaction of step (6) may be selected from the group consisting of dichloromethane, chloroform and mixtures thereof.
상기 단계 (7)에서는, 극성 비양성자성 용매 중에 합성된 펩타이드와 커플링 시약을 가하여 고리화 반응을 수행하여 폴리펩타이드 중간체를 제조한다.In step (7), a polypeptide intermediate is prepared by adding a coupling reagent and a coupling reagent in a polar aprotic solvent to perform a cyclization reaction.
상기 단계 (7)의 반응에 사용되는 커플링 시약은 1-히드록시-1H-벤조트리아졸/1,3-디이소프로필카보디이미드 또는 HATU (1-[비스(디메틸아미노)메틸렌]-1H-1,2,3-트리아졸로[4,5-b]-피리디늄 3-옥시드헥사플루오로포스페이트, 헥사플루오로포스페이트 아자벤조트리아졸 테트라메틸 유로늄)/N,N-디이소프로필에틸아민으로부터 선택될 수 있으나, 이들로 제한되는 것은 아니며 이러한 목적으로 당업계에 알려진 적합한 다른 커플링 시약들 또한 본 발명의 범위 내에서 사용 가능하다. The coupling reagent used in the reaction of step (7) is 1-hydroxy-1H-benzotriazole / 1,3-diisopropylcarbodiimide or HATU (1- [bis (dimethylamino) methylene] -1H -1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium) / N, N-diisopropylethyl Other coupling reagents known in the art for this purpose may also be used within the scope of the present invention, although it may be selected from amines.
또한, 본 발명의 신규한 폴리펩타이드 중간체를 사용한 생리활성 폴리펩타이드의 제조방법을 단계별로 보다 구체적으로 설명하면 다음과 같다.In addition, the method for producing a bioactive polypeptide using the novel polypeptide intermediate of the present invention will be described in more detail step by step.
상기 단계 (1)에서는, 상기의 방법으로 제조한 고리화된 펩타이드 화합물을 극성 비양성자성 용매 중에서 피페리딘 용액을 사용하여 보호기를 탈보호화시켜 탈보호화된 레진을 제조한다.In step (1), the deprotected resin is prepared by deprotecting the protecting group using the piperidine solution of the cyclized peptide compound prepared by the above method in a polar aprotic solvent.
상기 단계 (2)에서는, 극성 비양성자성 용매 중에 보호화된 아미노산, 1-히드록시-1H-벤조트리아졸 및 1,3-디이소프로필카보디이미드를 가하여 보호화된 아미노산을 활성화시킨다.In step (2), protected amino acids, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide are added in a polar aprotic solvent to activate the protected amino acids.
상기 단계 (3)에서는, 반응기 내 탈보호화된 수지에 활성화된 보호화된 아미노산 용액을 가하여 커플링 반응을 수행한다.In step (3), an activated protected amino acid solution is added to the deprotected resin in the reactor to carry out the coupling reaction.
상기 단계 (4)에서는, 펩타이드가 형성될 때까지 단계 (1)-(3)을 반복하여 수행한다. In step (4), steps (1)-(3) are repeated until a peptide is formed.
상기 단계 (4)의 반응은, 원하는 길이의 펩타이드가 형성될 때까지 1 내지 50번 반복하여 수행할 수 있고, 바람직하게는 1 내지 30번을 반복하여 수행할 수 있다.The reaction of step (4) may be performed repeatedly 1 to 50 times, preferably 1 to 30 times, until a peptide of a desired length is formed.
상기 단계 (5)에서는, 절단 칵테일을 사용하여 보호화된 레진을 탈보호화시키는 동시에 레진으로부터 원하는 펩타이드를 절단한다.In step (5) above, the cleavage cocktail is used to deprotect the protected resin while simultaneously cleaving the desired peptide from the resin.
상기 단계 (5)의 절단 칵테일은 트리플루오로아세트산 (TFA), 1종 이상의 스캐빈저 및 디클로로메탄의 용액을 포함할 수 있다. The cleavage cocktail of step (5) may comprise a solution of trifluoroacetic acid (TFA), one or more scavengers and dichloromethane.
상기 단계 (5)의 스캐빈저는 상기 스캐빈저는 트리이소프로필실란 (TIPS), 트리에틸실란 (TES), 페놀, 아니솔, 티오아니솔, 물, 에탄디티올 (EDT), 1-도데칸티올, 디티오트레이톨 (DTT) 및 인돌로 이루어진 군으로부터 선택될 수 있으나, 이들로 제한되는 것은 아니며 이러한 목적으로 당업계에 알려진 적합한 다른 스캐빈저들 또한 본 발명의 범위 내에서 사용 가능하다. The scavenger of step (5) is said scavenger is triisopropylsilane (TIPS), triethylsilane (TES), phenol, anisole, thioanisole, water, ethanedithiol (EDT), 1-dodecane Thiol, dithiothreitol (DTT) and indole may be selected from the group consisting of, but not limited to, other suitable scavengers known in the art for this purpose are also available within the scope of the present invention.
상기 단계 (6)에서는, 레진으로부터 절단 혼합물을 여과하는 단계를 포함하는 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염을 제조하는 방법을 제공한다.In step (6), there is provided a method of preparing a bioactive polypeptide and a pharmaceutically acceptable salt comprising filtering the cleavage mixture from the resin.
상술한 방법으로부터 본 발명의 일 구체예에서는 하기 화학식 2의 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염을 제조한다. In one embodiment of the present invention from the above-described method to prepare a bioactive polypeptide and a pharmaceutically acceptable salt of the formula (2).
[화학식 2][Formula 2]
Figure PCTKR2019008935-appb-img-000005
Figure PCTKR2019008935-appb-img-000005
: H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Ser-Lys-Tyr-Leu-Asp- Cyclo(-Glu-Lys-Arg-Ala-Lys)-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-NH 2 : H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Ser-Lys-Tyr-Leu-Asp- Cyclo (-Glu-Lys-Arg-Ala- Lys) -Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-NH 2
본 발명에서 제공하는 신규한 폴리펩타이드 중간체 및 이의 제조방법을 통하여 고순도의 의약품 중간체를 제공할 수 있고, 이를 이용하여 제조한 생리활성 폴리펩타이드도 순도가 높고, 정제가 용이하여 고품질의 의약품 제조에 사용이 가능하다. The novel polypeptide intermediate provided by the present invention and a pharmaceutical intermediate of high purity can be provided through the preparation method thereof, and the bioactive polypeptide prepared by using the same can also be used for the manufacture of high-quality pharmaceutical products with high purity and easy purification. This is possible.
이와 비교하여, 폴리펩타이드를 선형 합성한 후 고리화 반응을 수행하여 생리활성 폴리펩타이드를 제조할 수 있다. 이 경우, 16개의 아미노산 내지 30개의 아미노산을 순차적으로 선형 합성하고, 각각의 선형 합성된 폴리펩타이드 (16mer ~ 30mer)의 고리화 반응을 수행할 수 있다. 그러나, 이 경우 선형 합성된 폴리펩타이드의 길이가 증가함에 따라 순도가 급격하게 감소하는 경향을 보인다. 또한, 정제공정이 매우 어려워지고 그에 따라 수율이 급격하게 감소하는 경향을 보이게 된다.In comparison, a physiologically active polypeptide can be prepared by linear synthesis of the polypeptide followed by a cyclization reaction. In this case, linearly synthesize 16 to 30 amino acids sequentially and perform a cyclization reaction of each linear synthesized polypeptide (16mer to 30mer). In this case, however, the purity tends to decrease rapidly as the length of the linearly synthesized polypeptide increases. In addition, the purification process becomes very difficult and thus yields tend to decrease rapidly.
이와 같이 본 발명에 따른 신규한 폴리펩타이드 중간체 및 이의 제조방법을 통하여 제조한 생리활성 폴리펩타이드는 15mer까지의 선형 폴리펩타이드를 제조한 후 고리화 반응을 수행하고 나머지 아미노산을 추가로 합성하는 단계를 거치므로, 기존의 제조방법과 비교하여 고순도의 제품을 합성할 수 있는 장점을 가지고 있다. 또한, 본 발명의 제조방법은 합성 이후의 정제 공정이 용이할 뿐만 아니라, 이에 따라 최종 제조 수율이 매우 향상되어 상업화 생산에 적합한 효율적인 공정이라는 장점이 있다.As described above, the novel polypeptide intermediate and the physiologically active polypeptide prepared through the preparation method according to the present invention undergo a step of preparing a linear polypeptide up to 15mer, performing a cyclization reaction, and further synthesizing the remaining amino acids. Therefore, it has the advantage of synthesizing a high purity product compared to the existing manufacturing method. In addition, the manufacturing method of the present invention not only facilitates the purification process after synthesis, but also has the advantage that the final manufacturing yield is greatly improved and is an efficient process suitable for commercial production.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하여 설명한다. 다만 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 범주 및 기술사상 범위 내에서 다양한 변형 및 수정이 가능함은 당업자에게 있어 자명하다.Hereinafter, the present invention will be described with reference to preferred embodiments. However, the following examples are merely illustrative of the present invention, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope and spirit of the present invention.
본 명세서 전반을 통하여, 천연적으로 존재하는 아미노산에 대하여 통상적으로 1문자 또는 3문자 코드가 사용되고, Aib (α-아미노이소부티르산)과 같은 다른 아미노산에 대하여 일반적으로 허용되는 3문자 코드가 사용된다. 또한, 본 명세서에서 약어로 언급된 아미노산은 IUPAC-IUB 명명법에 따라 기재되었다.Throughout this specification, one or three letter codes are commonly used for naturally occurring amino acids, and three letter codes are generally accepted for other amino acids such as Aib (α-aminoisobutyric acid). In addition, amino acids referred to herein as abbreviations are described according to the IUPAC-IUB nomenclature.
알라닌 - Ala, A; 아르기닌 - Arg, R; 아스파라긴 - Asn, N;Alanine-Ala, A; Arginine-Arg, R; Asparagine-Asn, N;
아스파트산 - Asp, D; 시스테인 - Cys, C; 글루탐산 - Glu, E;Aspartic acid-Asp, D; Cysteine-Cys, C; Glutamic acid-Glu, E;
글루타민 - Gln, Q; 글리신 - Gly, G; 히스티딘 - His, H;Glutamine-Gln, Q; Glycine-Gly, G; Histidine-His, H;
이소루이신 - Ile, I; 루이신 - Leu, L; 라이신 - Lys, K;Isoleucine-Ile, I; Leucine-Leu, L; Lysine-Lys, K;
메티오닌 - Met, M; 페닐알라닌 - Phe, F; 프롤린 - Pro, P;Methionine-Met, M; Phenylalanine-Phe, F; Proline-Pro, P;
세린 - Ser, S; 트레오닌 - Thr, T; 트립토판 - Trp, W;Serine-Ser, S; Threonine-Thr, T; Tryptophan-Trp, W;
타이로신 - Tyr, Y; 발린 - Val, VTyrosine-Tyr, Y; Valine-Val, V
고체상 펩타이드 합성 방법(아미노산의 탈보호화 방법, 펩타이드를 수지로부터 절단하는 방법, 및 그것의 정제를 포함하는, SPPS 방법), 그뿐만 아니라 결과되는 펩타이드의 검출 및 특징화 방법(LCMS, MALDI, 및 UPLC 방법)과 관련된다.Solid phase peptide synthesis methods (including deprotection of amino acids, methods of cleaving peptides from resins, and SPPS methods, including purification thereof), as well as methods of detecting and characterizing the resulting peptides (LCMS, MALDI, and UPLC) Method).
사용되는 보호된 아미노산 유도체는 표준 Fmoc-아미노산이다. N-말단 아미노산은 알파 아미노기가 Boc으로 보호되었다 (예를 들어 N-말단에서 His를 갖는 펩타이드를 위한 Boc-His(Boc)-OH, 또는 Boc-His(Trt)-OH).Protected amino acid derivatives used are standard Fmoc-amino acids. N-terminal amino acids have an alpha amino group protected with Boc (eg Boc-His (Boc) -OH, or Boc-His (Trt) -OH for peptides having His at the N-terminus).
[수지 결합 펩타이드의 합성][Synthesis of Resin Binding Peptides]
실시예 1: 링크 아미드 MBHA 레진 준비Example 1: Link Amide MBHA Resin Preparation
용기에 링크 아미드 MBHA 레진 80.0g(0.31mmol/g)과 디메틸포름아미드 480㎖을 투입하고 15분 동안 교반한 후 디메틸포름아미드 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 상기 과정을 거쳐 링크 아미드 MBHA 레진을 준비하였다.80.0 g (0.31 mmol / g) of link amide MBHA resin and 480 ml of dimethylformamide were added to the vessel, stirred for 15 minutes, and filtered to remove dimethylformamide. This process was carried out twice. Through the above procedure, link amide MBHA resin was prepared.
실시예 2: 폴리펩타이드의 제조(1~15mer 합성)Example 2 Preparation of Polypeptides (1-15mer Synthesis)
합성 1) 레진-Cys(Trt)-Fmoc의 제조(폴리 1mer 합성)Synthesis 1) Preparation of Resin-Cys (Trt) -Fmoc (Poly 1mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 실시예 1에서 준비된 수지에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 링크 아미드 MBHA 레진을 얻었다. 480 ml of 20% piperidine was added to the resin prepared in Example 1, stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound Fmoc deprotected link amide MBHA resin was obtained.
단계 (2): Cys(Trt)-Fmoc (C)의 활성화Step (2): Activation of Cys (Trt) -Fmoc (C)
용기에 Cys(Trt)-Fmoc 43.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Cys(Trt)-Fmoc을 활성화 시켰다. 43.6 g of Cys (Trt) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above procedure, the target compound Cys (Trt) -Fmoc was activated.
단계 (3): 레진-Cys(Trt)-Fmoc의 제조(폴리 1mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Fmoc (Poly 1mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 링크 아미드 MBHA 레진에 단계 (2)에서 활성화시킨 Cys(Trt)-Fmoc을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 레진-Cys(Trt)-Fmoc을 얻었다. Cys (Trt) -Fmoc activated in step (2) was added to the Fmoc deprotected link amide MBHA resin prepared in step (1), stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Resin-Cys (Trt) -Fmoc, was obtained.
합성 2) 레진-Cys(Trt)-Thr(tBu)-Fmoc의 제조(폴리 2mer 합성)Synthesis 2) Preparation of Resin-Cys (Trt) -Thr (tBu) -Fmoc (Poly 2mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 1)에서 제조한 폴리펩타이드(폴리 2mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 레진-Cys(Trt) 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 2mer synthesis) prepared in Synthesis 1), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound, Fmoc deprotected resin-Cys (Trt) was obtained.
단계 (2): Thr(tBu)-Fmoc (T)의 활성화Step (2): Activation of Thr (tBu) -Fmoc (T)
용기에 Thr(tBu)-Fmoc 29.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Thr(tBu)-Fmoc 을 활성화 시켰다.29.6 g of Thr (tBu) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Thr (tBu) -Fmoc was activated.
단계 (3): 레진-Cys(Trt)-Thr(tBu)-Fmoc 의 제조(폴리 2mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Fmoc (Poly 2mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 레진-Cys(Trt)에 단계 (2)에서 활성화시킨 Thr(tBu)-Fmoc을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 레진-Cys(Trt)-Thr(tBu)-Fmoc(C)을 얻었다. Thr (tBu) -Fmoc activated in step (2) was added to the Fmoc deprotected resin-Cys (Trt) prepared in step (1), followed by stirring at room temperature for 3 hours or more, followed by filtration. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Resin-Cys (Trt) -Thr (tBu) -Fmoc (C) was obtained.
합성 3) 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc의 제조(폴리 3mer 합성)Synthesis 3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Fmoc (Poly 3mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 2)에서 제조한 폴리펩타이드(폴리 2mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 레진-Cys(Trt)-Thr(tBu) 을 얻었다. 480 mL of 20% piperidine was added to the polypeptide (poly 2mer synthesis) prepared in Synthesis 2), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound Fmoc deprotected resin-Cys (Trt) -Thr (tBu) was obtained.
단계 (2): Asn(Trt)-Fmoc (N)의 활성화Step (2): Activation of Asn (Trt) -Fmoc (N)
용기에 Asn(Trt)-Fmoc 44.4g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Asn(Trt)-Fmoc 을 활성화 시켰다.44.4 g of Asn (Trt) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Asn (Trt) -Fmoc, a target compound, was activated.
단계 (3): 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc 의 제조(폴리 3mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Fmoc (Poly 3mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 레진-Cys(Trt)-Thr(tBu)에 단계 (2)에서 활성화시킨 Asn(Trt)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc 을 얻었다.Asn (Trt) -Fmoc activated in step (2) was added to the Fmoc deprotected resin-Cys (Trt) -Thr (tBu) prepared in step (1), followed by stirring at room temperature for 3 hours or more, followed by filtration. . 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Fmoc, was obtained.
합성 4) 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc의 제조(폴리 4mer 합성)Synthesis 4) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc (Poly 4mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 3)에서 제조한 폴리펩타이드(폴리 3mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 레진-Cys(Trt)-Thr(tBu)-Asn(Trt) 을 얻었다. 480 mL of 20% piperidine was added to the polypeptide (poly 3mer synthesis) prepared in Synthesis 3), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound, Fmoc deprotected resin-Cys (Trt) -Thr (tBu) -Asn (Trt) was obtained.
단계 (2): Met-Fmoc (M)의 활성화Step (2): Activation of Met-Fmoc (M)
용기에 Met-Fmoc 27.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Met-Fmoc 을 활성화 시켰다.27.6 g of Met-Fmoc, 1H-benzotriazole, 1-hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Met-Fmoc, the target compound, was activated.
단계 (3): 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc 의 제조(폴리 4mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc (Poly 4mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)에 단계 (2)에서 활성화시킨 Met-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc 을 얻었다.After adding the activated Met-Fmoc in step (2) to the Fmoc deprotected resin-Cys (Trt) -Thr (tBu) -Asn (Trt) prepared in step (1) and stirred at room temperature for 3 hours or more. Filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc, was obtained.
합성 5) 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc의 제조(폴리 5mer 합성)Synthesis 5) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc (Poly 5mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 4)에서 제조한 폴리펩타이드(폴리 4mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 4mer synthesis) prepared in Synthesis 4), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Fmoc deprotected resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met, was obtained.
단계 (2): Leu-Fmoc (L)의 활성화Step (2): Activation of Leu-Fmoc (L)
용기에 Leu-Fmoc 26.3g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Leu-Fmoc 을 활성화 시켰다.26.3 g of Leu-Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Leu-Fmoc, the target compound, was activated.
단계 (3): 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc의 제조(폴리 5mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc (Poly 5mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 레진-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met에 단계 (2)에서 활성화시킨 Leu-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc 을 얻었다.Lemo-Fmoc activated in step (2) was added to the Fmoc deprotected resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met prepared in step (1), followed by stirring at room temperature for 3 hours or more. And then filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc was obtained.
합성 6) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc의 제조(폴리 6mer 합성)Synthesis 6) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc (Poly 6mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 5)에서 제조한 폴리펩타이드(폴리 5mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu 을 얻었다. 480 mL of 20% piperidine was added to the polypeptide (poly 5mer synthesis) prepared in Synthesis 5), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu, was obtained.
단계 (2): Trp(Boc)-Fmoc (W)의 활성화Step (2): Activation of Trp (Boc) -Fmoc (W)
용기에 Trp(Boc)-Fmoc 39.2g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Trp(Boc)-Fmoc 을 활성화 시켰다.39.2 g of Trp (Boc) -Fmoc, 1H-benzotriazole, 16.7 g, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Trp (Boc) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc 의 제조(폴리 6mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc (Poly 6mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu에 단계 (2)에서 활성화시킨 Trp(Boc)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc 을 얻었다.The Tmo (Boc) -Fmoc activated in step (2) was added to the Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu prepared in step (1). The mixture was stirred at least 3 hours and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc was obtained.
합성 7) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Fmoc의 제조 (폴리 7mer 합성)Synthesis 7) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc (Poly 7mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 6)에서 제조한 폴리펩타이드(폴리 6mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc) 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 6mer synthesis) prepared in Synthesis 6), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) was obtained.
단계 (2): Gln(Trt)-Fmoc (Q)의 활성화Step (2): Activation of Gln (Trt) -Fmoc (Q)
용기에 Gln(Trt)-Fmoc 45.4g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Gln(Trt)-Fmoc 을 활성화 시켰다.45.4 g of Gln (Trt) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Gln (Trt) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Fmoc 의 제조 (폴리 7mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc (Poly 7mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)에 단계 (2)에서 활성화시킨 Gln(Trt)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Fmoc 을 얻었다. Gln (Trt)-activated in step (2) to Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) prepared in step (1). Fmoc was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc was obtained.
합성 8) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Fmoc의 제조(폴리 8mer 합성)Synthesis 8) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Fmoc (Poly 8mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 7)에서 제조한 폴리펩타이드(폴리 7mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt) 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 7mer synthesis) prepared in Synthesis 7), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) was obtained through the above procedure.
단계 (2): Val-Fmoc (V)의 활성화Step (2): activation of Val-Fmoc (V)
용기에 Val-Fmoc 25.3g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Val-Fmoc 을 활성화 시켰다.25.3 g of Val-Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Val-Fmoc, the target compound, was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Fmoc 의 제조(폴리 8mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Fmoc (Poly 8mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)에 단계 (2)에서 활성화시킨 Val-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Fmoc을 얻었다.The Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) prepared in step (1) was activated in step (2). Val-Fmoc was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Fmoc was obtained.
합성 9) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Fmoc의 제조(폴리 9mer 합성)Synthesis 9) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Fmoc (Poly 9mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 8)에서 제조한 폴리펩타이드(폴리 8mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 8mer synthesis) prepared in Synthesis 8), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val was obtained.
단계 (2): Phe-Fmoc (F)의 활성화Step (2): Activation of Phe-Fmoc (F)
용기에 Phe-Fmoc 28.2g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Phe-Fmoc 을 활성화 시켰다.28.2 g of Phe-Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Phe-Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Fmoc 의 제조(폴리 9mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Fmoc (Poly 9mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val에 단계 (2)에서 활성화시킨 Phe-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val prepared in step (1) above in step (2) The activated Phe-Fmoc was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Fmoc was obtained.
합성 10) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Fmoc의 제조(폴리 10mer 합성)Synthesis 10) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Fmoc (Poly 10mer Synthesis) )
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 9)에서 제조한 폴리펩타이드(폴리 9mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 9mer synthesis) prepared in Synthesis 9), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe.
단계 (2): Glu(OtBu)-Fmoc (E)의 활성화Step (2): Activation of Glu (OtBu) -Fmoc (E)
용기에 Glu(OtBu)-Fmoc 31.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Glu(OtBu)-Fmoc 을 활성화 시켰다.31.6 g of Glu (OtBu) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Glu (OtBu) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Fmoc 의 제조(폴리 10mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Fmoc (Poly 10mer compound)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe에 단계 (2)에서 활성화시킨 Glu(OtBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe prepared in step (1) Glu (OtBu) -Fmoc activated at) was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Fmoc was obtained. .
합성 11) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Fmoc의 제조(폴리 11mer 합성)Synthesis 11) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Fmoc Manufacturing (Poly 11mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 10)에서 제조한 폴리펩타이드(폴리 10mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu) 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 10mer synthesis) prepared in Synthesis 10), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) Got.
단계 (2): Lys(Alloc)-Fmoc (K)의 활성화Step (2): Activation of Lys (Alloc) -Fmoc (K)
용기에 Lys(Alloc)-Fmoc 33.7g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Lys(Alloc)-Fmoc 을 활성화 시켰다.33.7 g of Lys (Alloc) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Lys (Alloc) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Fmoc 의 제조(폴리 11mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Fmoc (Poly 11mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)에 단계 (2)에서 활성화시킨 Lys(Alloc)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) Lys (Alloc) -Fmoc activated in step (2) was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) ) -Fmoc was obtained.
합성 12) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Fmoc의 제조(폴리 12mer 합성)Synthesis 12) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Fmoc (Poly 12mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 11)에서 제조한 폴리펩타이드(폴리 11mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc) 을 얻었다. 480 mL of 20% piperidine was added to the polypeptide (poly 11mer synthesis) prepared in Synthesis 11), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) was obtained.
단계 (2): Ala-Fmoc (A)의 활성화Step (2): Activation of Ala-Fmoc (A)
용기에 Ala-Fmoc 23.2g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Ala-Fmoc 을 활성화 시켰다.23.2 g of Ala-Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Ala-Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Fmoc 의 제조(폴리 12mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Fmoc (Poly 12mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)에 단계 (2)에서 활성화시킨 Ala-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Ala-Fmoc activated in step (2) was added to) -Lys (Alloc), and the mixture was stirred at room temperature for 3 hours or more and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) ) -Ala-Fmoc was obtained.
합성 13) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Fmoc의 제조(폴리 13mer 합성)Synthesis 13) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Fmoc (Poly 13mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 12)에서 제조한 폴리펩타이드(폴리 12mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 12mer synthesis) prepared in Synthesis 12), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala was obtained.
단계 (2): Arg(Pbf)-Fmoc (R)의 활성화Step (2): Activation of Arg (Pbf) -Fmoc (R)
용기에 Arg(Pbf)-Fmoc 48.3g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Arg(Pbf)-Fmoc 을 활성화 시켰다.48.3 g of Arg (Pbf) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above procedure, the target compound Arg (Pbf) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Arg(Pbf)-Fmoc 의 제조(폴리 13mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Fmoc (Poly 13mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala에 단계 (2)에서 활성화시킨 Arg(Pbf)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala- Arg(Pbf)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Arg (Pbf) -Fmoc activated in step (2) was added to) -Lys (Alloc) -Ala, and the mixture was stirred at room temperature for 3 hours or more and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) ) -Ala-Arg (Pbf) -Fmoc was obtained.
합성 14) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Fmoc의 제조(폴리 14mer 합성)Synthesis 14) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Fmoc (Poly 14mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 13)에서 제조한 폴리펩타이드(폴리 13mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf) 을 얻었다. 480 ml of 20% piperidine was added to the polypeptide (poly 13mer synthesis) prepared in Synthesis 13), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) was obtained.
단계 (2): Lys(Boc)-Fmoc (K)의 활성화Step (2): Activation of Lys (Boc) -Fmoc (K)
용기에 Lys(Boc)-Fmoc 34.9g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Lys(Boc)-Fmoc 을 활성화 시켰다.34.9 g of Lys (Boc) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Lys (Boc) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Arg(Pbf)-Lys(Boc)-Fmoc 의 제조(폴리 14mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Fmoc (Poly 14mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)에 단계 (2)에서 활성화시킨 Lys(Boc)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala- Arg(Pbf)-Lys(Boc)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Lys (Boc) -Fmoc activated in Step (2) was added to) -Lys (Alloc) -Ala-Arg (Pbf), and the mixture was stirred at room temperature for 3 hours or more and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) ) -Ala-Arg (Pbf) -Lys (Boc) -Fmoc was obtained.
합성 15) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Fmoc의 제조(폴리 15mer 합성)Synthesis 15) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Fmoc (Poly 15mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 14)에서 제조한 폴리펩타이드(폴리 14mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc) 을 얻었다. 480 mL of 20% piperidine was added to the polypeptide (poly 14mer synthesis) prepared in Synthesis 14), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) was obtained.
단계 (2): Glu(OAll)-Fmoc (E)의 활성화Step (2): Activation of Glu (OAll) -Fmoc (E)
용기에 Glu(OAll)-Fmoc 30.5g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Glu(OAll)-Fmoc 을 활성화 시켰다.Into the vessel, 30.5 g of Glu (OAll) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added and stirred to dissolve completely. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Glu (OAll) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Fmoc 의 제조(폴리 15mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Fmoc (Poly 15mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)에 단계 (2)에서 활성화시킨 Glu(OAll)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Glu (OAll) -Fmoc activated in step (2) was added to) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc), and the mixture was stirred at room temperature for 3 hours or more and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) ) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Fmoc was obtained.
실시예 3: 고리화 폴리펩타이드의 제조Example 3: Preparation of Cyclic Polypeptides
단계 (1): 부분 탈보호 제조 (Alloc/OAll)Step (1): Partial Deprotection Preparation (Alloc / OAll)
용기에 테트라키스팔라듐 11.5g과 디클로로메탄 800㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 N-메틸아닐린 78.6㎖와 페니실란 38.5㎖을 투입하고 5분 동안 교반시켰다. 상기 합성 15)에서 제조한 폴리펩타이드(폴리 15mer 합성)에 디클로로메탄 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 만들어 놓은 반응액을 여과된 수지에 투입하고 실온에서 5시간 동안 교반한 후 여과하였다. 여과된 수지에 디클로로메탄 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 5회 실시하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 2회 실시하였다.11.5 g of tetrakispalladium and 800 ml of dichloromethane were added to the vessel, followed by stirring to completely dissolve it. 78.6 ml of N-methylaniline and 38.5 ml of penicsilane were added to the dissolved reaction solution, and the mixture was stirred for 5 minutes. 480 ml of dichloromethane was added to the polypeptide prepared in Synthesis 15) (poly 15mer synthesis), followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. The prepared reaction solution was added to the filtered resin, stirred at room temperature for 5 hours, and filtered. 480 ml of dichloromethane was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out a total of five times. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out twice.
단계 (2)-1: 고리화 폴리펩타이드의 제조 (고리화 폴리 15mer 합성)Step (2) -1: Preparation of Cyclic Polypeptide (cyclic Poly 15mer Synthesis)
용기에 1H-벤조트라이아졸, 1-히드록시, 수화물 26.8g과 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 27.2㎖을 투입하고 상온에서 5분 동안 교반하였다. 상기 단계 (1)에서 제조한 폴리펩타이드(부분 탈보호 15mer)에 만들어 놓은 반응액을 투입하고 상온에서 5시간 동안 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 고리화 폴리펩타이드 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Fmoc 을 얻었다. 1H-benzotriazole, 1-hydroxy, hydrate 26.8 g, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to dissolve completely. 27.2 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 5 minutes. The reaction solution prepared in the polypeptide (partial deprotection 15mer) prepared in step (1) was added thereto, stirred at room temperature for 5 hours, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Cyclic polypeptide Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Fmoc was obtained.
단계 (2)-2: 고리화 폴리펩타이드의 제조 (고리화 폴리 15mer 합성)Step (2) -2: Preparation of Cyclic Polypeptide (cyclic Poly 15mer Synthesis)
용기에 HATU (1-[비스(디메틸아미노)메틸렌]-1H-1,2,3-트리아졸로[4,5-b]-피리디늄 3-옥시드 헥사플루오로포스페이트, 헥사플루오로포스페이트 아자벤조트리아졸 테트라메틸 유로늄,) 56.6g과 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 N,N-디이소프로필에틸아민 51.8㎖을 투입하고 상온에서 5분 동안 교반하였다. 상기 단계 (1)에서 제조한 폴리펩타이드(부분 탈보호 15mer)에 만들어 놓은 반응액을 투입하고 상온에서 3시간 동안 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 고리화 폴리펩타이드 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Fmoc 을 얻었다.HATU (1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate, hexafluorophosphate azabenzo in a container 56.6 g of triazole tetramethyl uronium,) and 480 ml of dimethylformamide were added and stirred to dissolve completely. 51.8 mL of N, N-diisopropylethylamine was added to the dissolved reaction solution, and the mixture was stirred at room temperature for 5 minutes. The reaction solution prepared in the polypeptide (partial deprotection 15mer) prepared in step (1) was added thereto, stirred at room temperature for 3 hours, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Cyclic polypeptide Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Fmoc was obtained.
실시예 4: 고리화 폴리펩타이드의 제조(16~30mer 합성)Example 4 Preparation of Cyclic Polypeptides (16-30mer Synthesis)
합성 1) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Fmoc 의 제조(고리화 폴리 16mer 합성)Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Fmoc (Synthetic Poly 16mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 실시예 3에서 제조한 고리화 폴리펩타이드(고리화 폴리 15mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu] 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (cyclic poly 15mer synthesis) prepared in Example 3, stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] was obtained.
단계 (2): Asp(OtBu)-Fmoc (D)의 활성화Step (2): Activation of Asp (OtBu) -Fmoc (D)
용기에 Asp(OtBu)-Fmoc 30.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Asp(OtBu)-Fmoc 을 활성화 시켰다. Into the vessel, 30.6 g of Asp (OtBu) -Fmoc, 1H-benzotriazole, 1-hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added and stirred to dissolve completely. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound, Asp (OtBu) -Fmoc, was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Fmoc 의 제조(고리화 폴리 16mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Fmoc (Synthetic Poly 16mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala- Arg(Pbf)-Lys(Boc)-Glu]에 단계 (2)에서 활성화시킨 Asp(OtBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Asp (OtBu) -Fmoc activated in step (2) was added to) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu], and the mixture was stirred at room temperature for 3 hours or more and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Fmoc was obtained.
합성 2) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Fmoc의 제조 (고리화 폴리 17mer 합성)Synthesis 2) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Fmoc (Synthetic Poly 17mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 1)에서 제조한 고리화 폴리펩타이드(고리화 폴리 16mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (cyclic poly 16mer synthesis) prepared in Synthesis 1), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) was obtained.
단계 (2): Leu-Fmoc (L)의 활성화Step (2): Activation of Leu-Fmoc (L)
용기에 Leu-Fmoc 26.3g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Leu-Fmoc 을 활성화 시켰다. 26.3 g of Leu-Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Leu-Fmoc, the target compound, was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Fmoc 의 제조(고리화 폴리 17mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Fmoc (Synthesized Poly 17mer)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)에 단계 (2)에서 활성화시킨 Leu-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Leu-Fmoc activated in step (2) was added to) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu), and the mixture was stirred at room temperature for 3 hours or more and filtered. . 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Fmoc was obtained.
합성 3) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc 의 제조(고리화 폴리 18mer 합성)Synthesis 3) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Fmoc (cyclic poly 18mer synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 2)에서 제조한 고리화 폴리펩타이드(고리화 폴리 17mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthetic poly 17mer) prepared in Synthesis 2), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu was obtained.
단계 (2): Tyr(tBu)-Fmoc (Y)의 활성화Step (2): Activation of Tyr (tBu) -Fmoc (Y)
용기에 Tyr(tBu)-Fmoc 34.2g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Tyr(tBu)-Fmoc 을 활성화 시켰다.34.2 g of Tyr (tBu) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Tyr (tBu) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc의 제조(고리화 폴리 18mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Fmoc (cyclic poly 18mer synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu 에 단계 (2)에서 활성화시킨 Tyr(tBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Add Tyr (tBu) -Fmoc activated in Step (2) to) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu for more than 3 hours at room temperature. After stirring, it was filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Fmoc was obtained.
합성 4) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc의 제조(고리화 폴리 19mer 합성)Synthesis 4) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Fmoc (cyclic poly 19mer synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 3)에서 제조한 고리화 폴리펩타이드(고리화 폴리 18mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthetic poly 18mer) prepared in Synthesis 3), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) was obtained.
단계 (2): Lys(Boc)-Fmoc (K)의 활성화Step (2): Activation of Lys (Boc) -Fmoc (K)
용기에 Lys(Boc)-Fmoc 34.9g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Lys(Boc)-Fmoc 을 활성화 시켰다.34.9 g of Lys (Boc) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Lys (Boc) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc의 제조(고리화 폴리 19mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Fmoc (cyclic poly 19mer synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)에 단계 (2)에서 활성화시킨 Lys(Boc)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Lys (Boc) -Fmoc activated in step (2) was added to) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu). After stirring at room temperature for 3 hours or more, the mixture was filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Fmoc was obtained.
합성 5) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc의 제조(고리화 폴리 20mer 합성)Synthesis 5) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Fmoc (synthesized poly 20mer)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 4)에서 제조한 고리화 폴리펩타이드(고리화 폴리 19mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthesized poly 19mer) prepared in Synthesis 4), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) was obtained.
단계 (2): Ser(tBu)-Fmoc (S)의 활성화Step (2): Activation of Ser (tBu) -Fmoc (S)
용기에 Ser(tBu)-Fmoc 28.5g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Ser(tBu)-Fmoc 을 활성화 시켰다.28.5 g of Ser (tBu) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Ser (tBu) -Fmoc, the target compound, was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc의 제조(고리화 폴리 20mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Fmoc (synthesized poly 20mer)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)에 단계 (2)에서 활성화시킨 Ser(tBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Ser (tBu) activated in step (2) to) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Fmoc was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Fmoc was obtained.
합성 6) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc의 제조(고리화 폴리 21mer 합성)Synthesis 6) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Fmoc (Synthesized Poly 21mer)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 5)에서 제조한 고리화 폴리펩타이드(고리화 폴리 20mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu) 을 얻었다. 480 mL of 20% piperidine was added to the cyclized polypeptide (synthesized poly 20mer) prepared in Synthesis 5), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) was obtained.
단계 (2): Tyr(tBu)-Fmoc (Y)의 활성화Step (2): Activation of Tyr (tBu) -Fmoc (Y)
용기에 Tyr(tBu)-Fmoc 34.2g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Tyr(tBu)-Fmoc 을 활성화 시켰다. 34.2 g of Tyr (tBu) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Tyr (tBu) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc의 제조(고리화 폴리 21mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Fmoc (cyclic poly 21mer synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)에 단계 (2)에서 활성화시킨 Tyr(tBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Activate in step (2) on) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) Tyr (tBu) -Fmoc was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Fmoc was obtained.
합성 7) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc의 제조(고리화 폴리 22mer 합성)Synthesis 7) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Fmoc Sum poly 22mer synthetic)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 6)에서 제조한 고리화 폴리펩타이드(고리화 폴리 21mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthesized poly 21mer) prepared in Synthesis 6), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) was obtained.
단계 (2): Asp(OtBu)-Fmoc (D)의 활성화Step (2): Activation of Asp (OtBu) -Fmoc (D)
용기에 Asp(OtBu)-Fmoc 30.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Asp(OtBu)-Fmoc 을 활성화 시켰다.Into the vessel, 30.6 g of Asp (OtBu) -Fmoc, 1H-benzotriazole, 1-hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added and stirred to dissolve completely. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound, Asp (OtBu) -Fmoc, was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc의 제조(고리화 폴리 22mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Fmoc (Compound Poly 22mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)에 단계 (2)에서 활성화시킨 Asp(OtBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) Step in) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) Asp (OtBu) -Fmoc activated in (2) was added thereto, stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Fmoc Got.
합성 8) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc의 제조(고리화 폴리 23mer 합성)Synthesis 8) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Fmoc (Synthetic Poly 23mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 7)에서 제조한 고리화 폴리펩타이드(고리화 폴리 22mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthesized poly 22mer) prepared in Synthesis 7), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) was obtained.
단계 (2): Ser(tBu)-Fmoc (S)의 활성화Step (2): Activation of Ser (tBu) -Fmoc (S)
용기에 Ser(tBu)-Fmoc 28.5g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Ser(tBu)-Fmoc 을 활성화 시켰다.28.5 g of Ser (tBu) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, Ser (tBu) -Fmoc, the target compound, was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc의 제조(고리화 폴리 23mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu ) -Fmoc Preparation (Synthetic Poly 23mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)에 단계 (2)에서 활성화시킨 Ser(tBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp Ser (tBu) -Fmoc activated in step (2) was added to (OtBu) and stirred at room temperature for 3 hours or more, followed by filtration. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Fmoc was obtained.
합성 9) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Fmoc의 제조(고리화 폴리 24mer 합성)Synthesis 9) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Thr (tBu) -Fmoc (Synthetic Poly 24mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 8)에서 제조한 고리화 폴리펩타이드(고리화 폴리 23mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthetic poly23mer) prepared in Synthesis 8), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) was obtained.
단계 (2): Thr(tBu)-Fmoc (T)의 활성화Step (2): Activation of Thr (tBu) -Fmoc (T)
용기에 Thr(tBu)-Fmoc 29.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Thr(tBu)-Fmoc 을 활성화 시켰다.29.6 g of Thr (tBu) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Thr (tBu) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)Fmoc의 제조(고리화 폴리 24mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) Fmoc (Synthetic Poly 24mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)에 단계 (2)에서 활성화시킨 Thr(tBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp Thr (tBu) -Fmoc activated in step (2) was added to (OtBu) -Ser (tBu), stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Fmoc was obtained.
합성 10) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc의 제조(고리화 폴리 25mer 합성)Synthesis 10) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Thr (tBu) -Phe-Fmoc (Synthetic Poly 25mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 9)에서 제조한 고리화 폴리펩타이드(고리화 폴리 24mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthetic poly24mer) prepared in Synthesis 9), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) -Thr (tBu) was obtained.
단계 (2): Phe-Fmoc (F)의 활성화Step (2): Activation of Phe-Fmoc (F)
용기에 Phe-Fmoc 28.8g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Phe-Fmoc 을 활성화 시켰다.28.8 g of Phe-Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Phe-Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc의 제조(고리화 폴리 25mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Fmoc (Synthetic Poly 25mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)에 단계 (2)에서 활성화시킨 Phe-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp Phe-Fmoc activated in step (2) was added to (OtBu) -Ser (tBu) -Thr (tBu), stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Fmoc was obtained.
합성 11) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc의 제조(고리화 폴리 26mer 합성)Synthesis 11) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Thr (tBu) -Phe-Thr (tBu) -Fmoc (Synthetic Poly 26mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 10)에서 제조한 고리화 폴리펩타이드(고리화 폴리 25mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthetic poly25mer) prepared in Synthesis 10), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) -Thr (tBu) -Phe was obtained.
단계 (2): Thr(tBu)-Fmoc (T)의 활성화Step (2): Activation of Thr (tBu) -Fmoc (T)
용기에 Thr(tBu)-Fmoc 29.6g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Thr(tBu)-Fmoc 을 활성화 시켰다.29.6 g of Thr (tBu) -Fmoc, 1H-benzotriazole, 16.7 hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Thr (tBu) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc의 제조(고리화 폴리 26mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Fmoc (cyclic poly 26mer synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe에 단계 (2)에서 활성화시킨 Thr(tBu)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp Thr (tBu) -Fmoc activated in step (2) was added to (OtBu) -Ser (tBu) -Thr (tBu) -Phe, and the mixture was stirred at room temperature for 3 hours or more and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Fmoc was obtained.
합성 12) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc의 제조(고리화 폴리 27mer 합성)Synthesis 12) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Thr (tBu) -Phe-Thr (tBu) -Gly-Fmoc (cyclic poly 27mer synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 11)에서 제조한 고리화 폴리펩타이드(고리화 폴리 26mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthetic poly 26mer) prepared in Synthesis 11), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) was obtained.
단계 (2): Gly-Fmoc (G)의 활성화Step (2): Activation of Gly-Fmoc (G)
용기에 Gly-Fmoc 22.1g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Gly-Fmoc 을 활성화 시켰다. 22.1 g of Gly-Fmoc, 1H-benzotriazole, 1-hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Gly-Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc의 제조(고리화 폴리 27mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Fmoc (Synthetic Poly 27mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)에 단계 (2)에서 활성화시킨 Gly-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc 을 얻었다.Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp Gly-Fmoc activated in step (2) was added to (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu), and the mixture was stirred at room temperature for 3 hours or more and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Fmoc was obtained.
합성 13) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc의 제조(고리화 폴리 28mer 합성)Synthesis 13) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Fmoc (cyclic poly 28mer synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 12)에서 제조한 고리화 폴리펩타이드(고리화 폴리 27mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthetic poly 27mer) prepared in Synthesis 12), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly was obtained.
단계 (2): Gln(Trt)-Fmoc (Q)의 활성화Step (2): Activation of Gln (Trt) -Fmoc (Q)
용기에 Gln(Trt)-Fmoc 45.4g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Gln(Trt)-Fmoc 을 활성화 시켰다.45.4 g of Gln (Trt) -Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Gln (Trt) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly- Gln(Trt)-Fmoc의 제조(고리화 폴리 28mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Fmoc (Synthetic Poly 28mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly에 단계 (2)에서 활성화시킨 Gln(Trt)-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly was added Gln (Trt) -Fmoc activated in step (2), stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Fmoc was obtained.
합성 14) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc 의 제조(고리화 폴리 29mer 합성)Synthesis 14) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-Fmoc (Synthesized Poly 29mer)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 13)에서 제조한 고리화 폴리펩타이드(고리화 폴리 28mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly- Gln(Trt) 을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthesized poly 28mer) prepared in Synthesis 13), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) was obtained.
단계 (2): Aib-Fmoc 의 활성화Step (2): Activation of Aib-Fmoc
용기에 Aib-Fmoc 24.2g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Aib-Fmoc 을 활성화 시켰다.24.2 g of Aib-Fmoc, 1H-benzotriazole, 1-hydroxy, 16.7 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound Aib-Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly- Gln(Trt)-Aib-Fmoc 의 제조(고리화 폴리 29mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-Fmoc (Synthetic Poly 29mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)에 단계 (2)에서 활성화시킨 Aib-Fmoc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp Aib-Fmoc activated in step (2) was added to (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) and stirred at room temperature for at least 3 hours, followed by filtration. It was. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-Fmoc was obtained.
합성 15) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc의 제조(고리화 폴리 30mer 합성)Synthesis 15) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala- Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu)- Preparation of Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-His (Trt) -Boc (cyclic poly 30mer synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 14)에서 제조한 고리화 폴리펩타이드(고리화 폴리 29mer 합성)에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib을 얻었다. 480 ml of 20% piperidine was added to the cyclized polypeptide (synthesized poly29mer) prepared in Synthesis 14), stirred for 20 minutes, and filtered to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib was obtained.
단계 (2): His(Trt)-Boc (H)의 활성화Step (2): Activation of His (Trt) -Boc (H)
용기에 His(Trt)-Boc 37.0g과 1H-벤조트라이아졸, 1-히드록시, 수화물 16.7g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 15.5㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 His(Trt)-Boc 을 활성화 시켰다.37.0 g of His (Trt) -Boc, 1H-benzotriazole, 1-hydroxy, hydrate 16.7 g, and 480 ml of dimethylformamide were added to the vessel, followed by stirring to completely dissolve it. 15.5 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above process, the target compound His (Trt) -Boc was activated.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly- Gln(Trt)-Aib-His(Trt)-Boc의 제조(고리화 폴리 30mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -cyclo- [Lys- Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-His (Trt) -Boc (cyclic poly 30mer synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib에 단계 (2)에서 활성화시킨 His(Trt)-Boc 을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc 을 얻었다. Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) prepared in step (1) ) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp His (Trt) -Boc activated in step (2) was added to (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib for 3 hours at room temperature. The mixture was stirred and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [ Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-His (Trt) -Boc was obtained.
실시예 5: NH2CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu]-Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib- His-NH2, TFA 의 제조 (보호기 및 레진 절단, cleavage)Example 5: NH 2 CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu] -Asp-Leu-Tyr-Lys-Ser- Preparation of Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-NH2, TFA (protector and resin cleavage, cleavage)
용기 2에 질소 분위기하에서 트리플루오로아세트산 165㎖와 페놀 10㎖, 증류수 10㎖, 티오아니솔 10㎖, 1,2-에테인디티올 5㎖를 투입하고 10분 동안 교반하였다. 용기 1에 건조된 고리화 폴리펩타이드 30mer 20g을 투입하고 용기 1에 준비된 반응액을 투입한 후 1시간 30분 동안 실온에서 교반하였다. 용기 3에 메틸 삼차 뷰틸 에터 3.0L를 투입하고 질소 분위기하에 0~1℃로 냉각하였다. 용기 1의 반응액 온도를 5℃로 냉각하고 용기 3의 냉각된 반응물 메틸 삼차 뷰틸 에터 2.0L를 취하여 투입한 후 10분 동안 교반하였다. 반응액을 여과하고 냉각된 메틸 삼차 뷰틸 에터 400㎖로 2번 세척한다. 여과기의 결정을 질소 분위기하에서 10분 동안 건조하였다. 용기 1에 건조된 결정을 투입하고 증류수 400㎖를 투입한 후 실온에서 10분 동안 교반하였다. 반응물을 여과하고 증류수 600㎖로 세척하여 목적 화합물인 NH 2CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo-[Lys-Ala-Arg-Lys-Glu]-Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib- His-NH 2, TFA 을 얻었다.In vessel 2, 165 ml of trifluoroacetic acid, 10 ml of phenol, 10 ml of distilled water, 10 ml of thioanisole, and 5 ml of 1,2-ethanedithiol were added to the vessel 2 and stirred for 10 minutes. 20 g of the dried cyclocyclized polypeptide 30mer was added to the vessel 1, and the prepared reaction solution was added to the vessel 1, followed by stirring at room temperature for 1 hour 30 minutes. 3.0 L of methyl tertiary butyl ether was put into the container 3, and it cooled to 0-1 degreeC under nitrogen atmosphere. The reaction liquid temperature of the vessel 1 was cooled to 5 ° C., 2.0 L of the cooled reactant methyl tertiary butyl ether of the vessel 3 was added thereto, and stirred for 10 minutes. The reaction solution is filtered and washed twice with 400 ml of cooled methyl tertiary butyl ether. Crystals of the filter were dried under nitrogen atmosphere for 10 minutes. Dried crystals were added to the vessel 1, 400 ml of distilled water was added thereto, followed by stirring at room temperature for 10 minutes. The reaction was filtered and washed with distilled water 600ml to give the target compound NH 2 CO-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu] -Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-NH 2 , TFA was obtained.
비교예 1: Rink amide MBHA resin 준비Comparative Example 1: Preparation of Rink amide MBHA resin
용기에 Rink amide MBHA resin 80.0g(0.31mmol/g)과 디메틸포름아미드 480㎖을 투입하고 15분 동안 교반한 후 디메틸포름아미드 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 상기 과정을 거쳐 Rink amide MBHA resin을 준비하였다.Rink amide MBHA resin 80.0g (0.31 mmol / g) and 480 ml of dimethylformamide were added to the vessel, stirred for 15 minutes, and filtered to remove dimethylformamide. This process was carried out twice. Rink amide MBHA resin was prepared by the above procedure.
비교예 2: 폴리펩타이드의 제조(1~30mer 합성)Comparative Example 2: Preparation of Polypeptides (1-30mer Synthesis)
합성 1) Resin-Cys(Trt)-Fmoc의 제조(폴리 1mer 합성)Synthesis 1) Preparation of Resin-Cys (Trt) -Fmoc (Polymer 1mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 비교예 1에서 준비된 수지에 20% 피페리딘 480㎖을 투입하고 20분 동안 교반한 후 20% 피페리딘 제거를 위해 여과하였다. 이 과정을 총 2회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Fmoc 탈보호화된 Rink amide MBHA resin을 얻었다. 480 mL of 20% piperidine was added to the resin prepared in Comparative Example 1, followed by stirring for 20 minutes, followed by filtration to remove 20% piperidine. This process was carried out twice. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, a target compound, Fmoc deprotected Rink amide MBHA resin was obtained.
단계 (2): Cys(Trt)-Fmoc (C)의 활성화Step (2): Activation of Cys (Trt) -Fmoc (C)
용기에 Cys(Trt)-Fmoc 29.1g과 1H-벤조트라이아졸, 1-히드록시, 수화물 13.4g, 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 1,3-디이소프로필카보디이미드 11.6㎖을 투입하고 상온에서 30분 동안 교반하였다. 상기 과정을 거쳐 목적화합물인 Cys(Trt)-Fmoc을 활성화 시켰다.29.1 g of Cys (Trt) -Fmoc, 1H-benzotriazole, 13.4 hydroxy, 13.4 g of hydrate, and 480 ml of dimethylformamide were added to the vessel, followed by complete dissolution. 11.6 ml of 1,3-diisopropylcarbodiimide was added to the dissolved reaction solution, followed by stirring at room temperature for 30 minutes. Through the above procedure, the target compound Cys (Trt) -Fmoc was activated.
단계 (3): Resin-Cys(Trt)-Fmoc의 제조(폴리 1mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Fmoc (Poly 1mer Synthesis)
상기 단계 (1)에서 제조된 Fmoc 탈보호화된 Rink amide MBHA resin에 단계 (2)에서 활성화시킨 Cys(Trt)-Fmoc을 투입하여 상온에서 3시간 이상 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 Resin-Cys(Trt)-Fmoc을 얻었다.Cys (Trt) -Fmoc activated in step (2) was added to the Fmoc deprotected Rink amide MBHA resin prepared in step (1), stirred at room temperature for 3 hours or more, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Through the above procedure, the target compound Resin-Cys (Trt) -Fmoc was obtained.
합성 2) Resin-Cys(Trt)-Thr(tBu)-Fmoc의 제조(폴리 2mer 합성)Synthesis 2) Preparation of Resin-Cys (Trt) -Thr (tBu) -Fmoc (Poly 2mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 1)에서 제조한 폴리펩타이드(폴리 1mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt) 을 얻었다. Producing the Fmoc deprotected Resin-Cys (Trt) as a target compound was carried out in the same manner as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 1).
단계 (2): Thr(tBu)-Fmoc (T)의 활성화Step (2): Activation of Thr (tBu) -Fmoc (T)
Thr(tBu)-Fmoc 19.7g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Thr(tBu)-Fmoc 을 활성화 시켰다.Thr (tBu) -Fmoc 19.7g Synthesis 1) In the same manner as in step (2) to activate the target compound Thr (tBu) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Fmoc 의 제조(폴리 2mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Fmoc (Poly 2mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Fmoc(C)을 얻었다.Proceed in the same manner as step (3) of the synthesis 1) to obtain the target compound Resin-Cys (Trt) -Thr (tBu) -Fmoc (C).
합성 3) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc의 제조(폴리 3mer 합성)Synthesis 3) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Fmoc (Poly 3mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 2)에서 제조한 폴리펩타이드(폴리 2mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu) 을 얻었다. Producing the Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) as a target compound in the same manner as in step (1) of Synthesis 1) using the polypeptide (poly 2mer synthesis) prepared in Synthesis 2).
단계 (2): Asn(Trt)-Fmoc (N)의 활성화Step (2): Activation of Asn (Trt) -Fmoc (N)
Asn(Trt)-Fmoc 29.6g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Asn(Trt)-Fmoc 을 활성화 시켰다.Asn (Trt) -Fmoc 29.6g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Asn (Trt) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc 의 제조(폴리 3mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Fmoc (Poly 3mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Fmoc 을 얻었다.Proceed as in step (3) of the synthesis 1) to obtain the target compound Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Fmoc.
합성 4) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc의 제조(폴리 4mer 합성)Synthesis 4) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc (Poly 4mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 3)에서 제조한 폴리펩타이드(폴리 3mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 3). Trt) was obtained.
단계 (2): Met-Fmoc (M)의 활성화Step (2): Activation of Met-Fmoc (M)
Met-Fmoc 18.4g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Met-Fmoc 을 활성화 시켰다.Met-Fmoc 18.4g was carried out in the same manner as in step (2) of Synthesis 1) to activate Met-Fmoc as a target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc 의 제조(폴리 4mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc (Poly 4mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of the synthesis 1) to obtain the target compound Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Fmoc.
합성 5) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc의 제조(폴리 5mer 합성)Synthesis 5) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc (Poly 5mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 4)에서 제조한 폴리펩타이드(폴리 4mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 4). Trt) -Met was obtained.
단계 (2): Leu-Fmoc (L)의 활성화Step (2): Activation of Leu-Fmoc (L)
Leu-Fmoc 17.5g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Leu-Fmoc 을 활성화 시켰다.Leu-Fmoc 17.5g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Leu-Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc 의 제조(폴리 5mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc (Poly 5mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Fmoc 을 얻었다.Proceed in the same manner as step (3) of Synthesis 1) to obtain the target compound Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Fmoc.
합성 6) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc의 제조(폴리 6mer 합성)Synthesis 6) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc (Poly 6mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 5)에서 제조한 폴리펩타이드(폴리 5mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 5). Trt) -Met-Leu was obtained.
단계 (2): Trp(Boc)-Fmoc (W)의 활성화Step (2): Activation of Trp (Boc) -Fmoc (W)
Trp(Boc)-Fmoc 26.1g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Trp(Boc)-Fmoc 을 활성화 시켰다.Trp (Boc) -Fmoc 26.1g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Trp (Boc) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc 의 제조(폴리 6mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc (Poly 6mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of the synthesis 1) to obtain the target compound Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Fmoc.
합성 7) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Fmoc의 제조(폴리 7mer 합성)Synthesis 7) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc (Poly 7mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 6)에서 제조한 폴리펩타이드(폴리 6mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc) 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (target compound) in the same manner as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 6). Trt) -Met-Leu-Trp (Boc) was obtained.
단계 (2): Gln(Trt)-Fmoc (Q)의 활성화Step (2): Activation of Gln (Trt) -Fmoc (Q)
Gln(Trt)-Fmoc 30.3g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Gln(Trt)-Fmoc 을 활성화 시켰다.Gln (Trt) -Fmoc 30.3g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Gln (Trt) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Fmoc 의 제조(폴리 7mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc (Poly 7mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Fmoc 을 얻었다.Proceed in the same manner as step (3) of Synthesis 1) to the target compound Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Fmoc Got it.
합성 8) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Fmoc의 제조(폴리 8mer 합성)Synthesis 8) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Fmoc (Poly 8mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 7)에서 제조한 폴리펩타이드(폴리 7mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt) 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (target compound) in the same manner as step (1) of Synthesis 1) with the polypeptide (poly 7mer synthesis) prepared in Synthesis 7). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) was obtained.
단계 (2): Val-Fmoc (V)의 활성화Step (2): activation of Val-Fmoc (V)
용기에 Val-Fmoc 16.8g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Val-Fmoc 을 활성화 시켰다.Val-Fmoc 16.8g into the container was carried out in the same manner as in step (2) of synthesis 1) to activate the target compound Val-Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Fmoc 의 제조(폴리 8mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Fmoc (Poly 8mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Fmoc was obtained.
합성 9) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Fmoc의 제조(폴리 9mer 합성)Synthesis 9) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Fmoc (Poly 9mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 8)에서 제조한 폴리펩타이드(폴리 8mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 8). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val was obtained.
단계 (2): Phe-Fmoc (F)의 활성화Step (2): Activation of Phe-Fmoc (F)
Phe-Fmoc 19.2g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Phe-Fmoc 을 활성화 시켰다.Phe-Fmoc 19.2g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Phe-Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Fmoc 의 제조(폴리 9mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Fmoc (Poly 9mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Fmoc was obtained.
합성 10) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Fmoc의 제조(폴리 10mer 합성)Synthesis 10) Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Fmoc (Poly 10mer Synthesis) )
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 9)에서 제조한 폴리펩타이드(폴리 9mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 9). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe was obtained.
단계 (2): Glu(OtBu)-Fmoc (E)의 활성화Step (2): Activation of Glu (OtBu) -Fmoc (E)
Glu(OtBu)-Fmoc 21.1g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Glu(OtBu)-Fmoc 을 활성화 시켰다.Glu (OtBu) -Fmoc 21.1g Synthesis 1) in the same manner as in step (2) to activate the target compound Glu (OtBu) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Fmoc 의 제조(폴리 10mer 합성)Step (3): Preparation of Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Fmoc (Poly 10mer compound)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Fmoc was obtained.
합성 11) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Fmoc의 제조(폴리 11mer 합성)Synthesis 11) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Fmoc Manufacturing (Poly 11mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 10)에서 제조한 폴리펩타이드(폴리 10mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) was obtained.
단계 (2): Lys(Alloc)-Fmoc (K)의 활성화Step (2): Activation of Lys (Alloc) -Fmoc (K)
Lys(Alloc)-Fmoc 22.4g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Lys(Alloc)-Fmoc 을 활성화 시켰다.Lys (Alloc) -Fmoc 22.4g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Lys (Alloc) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Fmoc 의 제조(폴리 11mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Fmoc (Poly 11mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Fmoc was obtained.
합성 12) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Fmoc의 제조(폴리 12mer 합성)Synthesis 12) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Fmoc (Poly 12mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 11)에서 제조한 폴리펩타이드(폴리 11mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 11). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) was obtained.
단계 (2): Ala-Fmoc (A)의 활성화Step (2): Activation of Ala-Fmoc (A)
Ala-Fmoc 15.4g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Ala-Fmoc 을 활성화 시켰다.Ala-Fmoc 15.4g was activated in the same manner as in step (2) of Synthesis 1) to activate Ala-Fmoc, the target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Fmoc 의 제조(폴리 12mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Fmoc (Poly 12mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Fmoc was obtained.
합성 13) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Fmoc의 제조(폴리 13mer 합성)Synthesis 13) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Fmoc (Poly 13mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 12)에서 제조한 폴리펩타이드(폴리 12mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 12). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala was obtained.
단계 (2): Arg(Pbf)-Fmoc (R)의 활성화Step (2): Activation of Arg (Pbf) -Fmoc (R)
Arg(Pbf)-Fmoc 32.2g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Arg(Pbf)-Fmoc 을 활성화 시켰다.Arg (Pbf) -Fmoc 32.2g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Arg (Pbf) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Arg(Pbf)-Fmoc 의 제조(폴리 13mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Fmoc (Poly 13mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala- Arg(Pbf)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Fmoc was obtained.
합성 14) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Fmoc의 제조(폴리 14mer 합성)Synthesis 14) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Fmoc (Poly 14mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 13)에서 제조한 폴리펩타이드(폴리 13mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 13). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) was obtained.
단계 (2): Lys(Boc)-Fmoc (K)의 활성화Step (2): Activation of Lys (Boc) -Fmoc (K)
Lys(Boc)-Fmoc 23.2g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Lys(Boc)-Fmoc 을 활성화 시켰다.Lys (Boc) -Fmoc 23.2g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Lys (Boc) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Arg(Pbf)-Lys(Boc)-Fmoc 의 제조(폴리 14mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Fmoc (Poly 14mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala- Arg(Pbf)-Lys(Boc)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Fmoc was obtained.
합성 15) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Fmoc의 제조(폴리 15mer 합성)Synthesis 15) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Fmoc (Poly 15mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 14)에서 제조한 폴리펩타이드(폴리 14mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 14). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) was obtained.
단계 (2): Glu(OAll)-Fmoc (E)의 활성화Step (2): Activation of Glu (OAll) -Fmoc (E)
Glu(OAll)-Fmoc 20.3g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Glu(OAll)-Fmoc 을 활성화 시켰다.Glu (OAll) -Fmoc 20.3g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Glu (OAll) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)- Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Fmoc 의 제조(폴리 15mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Fmoc (Poly 15mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Fmoc was obtained.
합성 16) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Fmoc 의 제조(폴리 16mer 합성)Synthesis 16) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Fmoc (Poly 16mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 15)에서 제조한 폴리펩타이드(폴리 15mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 15). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll).
단계 (2): Asp(OtBu)-Fmoc (D)의 활성화Step (2): Activation of Asp (OtBu) -Fmoc (D)
Asp(OtBu)-Fmoc 20.4g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Asp(OtBu)-Fmoc 을 활성화 시켰다.Asp (OtBu) -Fmoc 20.4g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Asp (OtBu) -Fmoc as a target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Fmoc 의 제조(폴리 16mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Fmoc (Poly 16mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Fmoc was obtained.
합성 17) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Fmoc 의 제조(폴리 17mer 합성)Synthesis 17) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Fmoc (Poly 17mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 16)에서 제조한 폴리펩타이드(폴리 16mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 16). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) was obtained.
단계 (2): Leu-Fmoc (L)의 활성화Step (2): Activation of Leu-Fmoc (L)
Leu-Fmoc 17.5g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Leu-Fmoc 을 활성화 시켰다.Leu-Fmoc 17.5g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Leu-Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Fmoc 의 제조(폴리 17mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Fmoc (Poly 17mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Fmoc was obtained.
합성 18) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc 의 제조(폴리 18mer 합성)Synthesis 18) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Fmoc (Poly 18mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 17)에서 제조한 폴리펩타이드(폴리 17mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 17). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu was obtained.
단계 (2): Tyr(tBu)-Fmoc (Y)의 활성화Step (2): Activation of Tyr (tBu) -Fmoc (Y)
Tyr(tBu)-Fmoc 22.8g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Tyr(tBu)-Fmoc 을 활성화 시켰다.Tyr (tBu) -Fmoc 22.8g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Tyr (tBu) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc 의 제조(폴리 18mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Fmoc (Poly 18mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Fmoc was obtained.
합성 19) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc 의 제조(폴리 19mer 합성)Synthesis 19) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Fmoc (Poly 19mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 18)에서 제조한 폴리펩타이드(폴리 18mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu) 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 18). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) was obtained.
단계 (2): Lys(Boc)-Fmoc (K)의 활성화Step (2): Activation of Lys (Boc) -Fmoc (K)
Lys(Boc)-Fmoc 23.2g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Lys(Boc)-Fmoc 을 활성화 시켰다.Lys (Boc) -Fmoc 23.2g Synthesis 1) was carried out in the same manner as in step (2) to activate the target compound Lys (Boc) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc 의 제조(폴리 19mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Fmoc (Poly 19mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Fmoc was obtained.
합성 20) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc 의 제조(폴리 20mer 합성)Synthesis 20) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Fmoc (Poly 20mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 19)에서 제조한 폴리펩타이드(폴리 19mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc) 을 얻었다. Producing the Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (Fmoc) as a target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 19). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) was obtained.
단계 (2): Ser(tBu)-Fmoc (S)의 활성화Step (2): Activation of Ser (tBu) -Fmoc (S)
Ser(tBu)-Fmoc 19.0g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Ser(tBu)-Fmoc 을 활성화 시켰다.Ser (tBu) -Fmoc 19.0g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Ser (tBu) -Fmoc as a target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc 의 제조(폴리 20mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Fmoc (Poly 20mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Fmoc was obtained.
합성 21) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc 의 제조(폴리 21mer 합성)Synthesis 21) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys-Ala-Arg (Pbf Preparation of) -Lys (Boc) -Glu-Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Fmoc (Poly 21mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 20)에서 제조한 폴리펩타이드(폴리 20mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu) 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn (target compound) in the same manner as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 20). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) was obtained.
단계 (2): Tyr(tBu)-Fmoc (Y)의 활성화Step (2): Activation of Tyr (tBu) -Fmoc (Y)
Tyr(tBu)-Fmoc 22.8g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Tyr(tBu)-Fmoc 을 활성화 시켰다.Tyr (tBu) -Fmoc 22.8g was synthesized in the same manner as in step (2) of Synthesis 1) to activate the target compound Tyr (tBu) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc 의 제조(폴리 21mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Preparation of Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Fmoc (Poly 21mer synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Fmoc was obtained.
합성 22) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc 의 제조(폴리 22mer 합성)Synthesis 22) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Preparation of Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Fmoc (Poly 22mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 21)에서 제조한 폴리펩타이드(폴리 21mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 21). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) was obtained.
단계 (2): Asp(OtBu)-Fmoc (D)의 활성화Step (2): Activation of Asp (OtBu) -Fmoc (D)
Asp(OtBu)-Fmoc 20.4g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Asp(OtBu)-Fmoc 을 활성화 시켰다.Asp (OtBu) -Fmoc 20.4g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Asp (OtBu) -Fmoc as a target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc 의 제조(폴리 22mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Fmoc Preparation (Poly 22mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Fmoc was obtained.
합성 23) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc 의 제조(폴리 23mer 합성)Synthesis 23) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu ) -Fmoc Preparation (Poly 23mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 22)에서 제조한 폴리펩타이드(폴리 22mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu) 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 22). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) was obtained.
단계 (2): Ser(tBu)-Fmoc (S)의 활성화Step (2): Activation of Ser (tBu) -Fmoc (S)
Ser(tBu)-Fmoc 19.0g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Ser(tBu)-Fmoc 을 활성화 시켰다.Ser (tBu) -Fmoc 19.0g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Ser (tBu) -Fmoc as a target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc 의 제조(폴리 23mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Fmoc (Poly 23mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys-(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys- (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu ) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Fmoc was obtained.
합성 24) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Fmoc 의 제조(폴리 24mer 합성)Synthesis 24) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Fmoc (Poly 24mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 23)에서 제조한 폴리펩타이드(폴리 23mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu) 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 23). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) was obtained.
단계 (2): Thr(tBu)-Fmoc (T)의 활성화Step (2): Activation of Thr (tBu) -Fmoc (T)
Thr(tBu)-Fmoc 19.7g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Thr(tBu)-Fmoc 을 활성화 시켰다.Thr (tBu) -Fmoc 19.7g Synthesis 1) In the same manner as in step (2) to activate the target compound Thr (tBu) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)Fmoc 의 제조(폴리 24mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Thr (tBu) Fmoc (Poly 24mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Fmoc was obtained.
합성 25) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc 의 제조(폴리 25mer 합성)Synthesis 25) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Fmoc (Poly 25mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 24)에서 제조한 폴리펩타이드(폴리 24mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu) 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 24). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) was obtained.
단계 (2): Phe-Fmoc (F)의 활성화Step (2): Activation of Phe-Fmoc (F)
Phe-Fmoc 19.2g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Phe-Fmoc 을 활성화 시켰다.Phe-Fmoc 19.2g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Phe-Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc 의 제조(폴리 25mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Thr (tBu) -Phe-Fmoc (Poly 25mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Fmoc was obtained.
합성 26) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc 의 제조(폴리 26mer 합성)Synthesis 26) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Fmoc (Poly 26mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 25)에서 제조한 폴리펩타이드(폴리 25mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 25). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe was obtained.
단계 (2): Thr(tBu)-Fmoc (T)의 활성화Step (2): Activation of Thr (tBu) -Fmoc (T)
Thr(tBu)-Fmoc 19.7g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Thr(tBu)-Fmoc 을 활성화 시켰다.Thr (tBu) -Fmoc 19.7g Synthesis 1) In the same manner as in step (2) to activate the target compound Thr (tBu) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc 의 제조(폴리 26mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Thr (tBu) -Phe-Thr (tBu) -Fmoc (Poly 26mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Fmoc was obtained.
합성 27) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc 의 제조(폴리 27mer 합성)Synthesis 27) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Fmoc (Poly 27mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 26)에서 제조한 폴리펩타이드(폴리 26mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 26). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) was obtained.
단계 (2): Gly-Fmoc (G)의 활성화Step (2): Activation of Gly-Fmoc (G)
Gly-Fmoc 14.7g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Gly-Fmoc 을 활성화 시켰다.Gly-Fmoc 14.7g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Gly-Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc 의 제조(폴리 27mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Fmoc (Poly 27mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Fmoc was obtained.
합성 28) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc 의 제조(고리화 폴리 28mer 합성)Synthesis 28) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Fmoc (cyclic poly 28mer synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 27)에서 제조한 폴리펩타이드(폴리 27mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly 을 얻었다. Promote Fmoc-deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () with the polypeptide prepared in Synthesis 27) in the same manner as in step (1) of Synthesis 1). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly .
단계 (2): Gln(Trt)-Fmoc (Q)의 활성화Step (2): Activation of Gln (Trt) -Fmoc (Q)
Gln(Trt)-Fmoc 30.3g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Gln(Trt)-Fmoc 을 활성화 시켰다.Gln (Trt) -Fmoc 30.3g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Gln (Trt) -Fmoc.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly- Gln(Trt)-Fmoc 의 제조(폴리 28mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Fmoc (Poly 28mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Fmoc was obtained.
합성 29) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc 의 제조(폴리 29mer 합성)Synthesis 29) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-Fmoc (Poly 29mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 28)에서 제조한 폴리펩타이드(폴리 28mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt) 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as the target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 28). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) was obtained.
단계 (2): Aib-Fmoc 의 활성화Step (2): Activation of Aib-Fmoc
Aib-Fmoc 16.1g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 Aib-Fmoc 을 활성화 시켰다.Aib-Fmoc 16.1g was synthesized in the same manner as in step (2) of Synthesis 1) to activate Aib-Fmoc as a target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly- Gln(Trt)-Aib-Fmoc 의 제조(폴리 29mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-Fmoc (Poly 29mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-Fmoc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-Fmoc was obtained.
합성 30) Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe- Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc 의 제조(폴리 30mer 합성)Synthesis 30) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala- Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu Preparation of) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-His (Trt) -Boc (Poly 30mer Synthesis)
단계 (1): Fmoc 탈보호 제조Step (1): Fmoc Deprotection Preparation
상기 합성 29)에서 제조한 폴리펩타이드(폴리 29mer 합성)로 합성 1)의 단계 (1)과 동일한 방법으로 진행하여 목적화합물인 Fmoc 탈보호화된 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib 을 얻었다. Promote Fmoc deprotected Resin-Cys (Trt) -Thr (tBu) -Asn () as a target compound by the same method as in step (1) of Synthesis 1) using the polypeptide prepared in Synthesis 29). Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp ( OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib was obtained.
단계 (2): His(Trt)-Boc (H)의 활성화Step (2): Activation of His (Trt) -Boc (H)
His(Trt)-Boc 30.7g으로 합성 1)의 단계 (2)와 동일한 방법으로 진행하여 목적화합물인 His(Trt)-Boc 을 활성화 시켰다.His (Trt) -Boc 30.7g was synthesized in the same manner as in step (2) of Synthesis 1) to activate His (Trt) -Boc as a target compound.
단계 (3): Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)- Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly- Gln(Trt)-Aib-His(Trt)-Boc 의 제조(폴리 30mer 합성)Step (3): Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt)-Val-Phe-Glu (OtBu) -Lys (Alloc)- Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser Preparation of (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-His (Trt) -Boc (Poly 30mer Synthesis)
합성 1)의 단계 (3)과 동일한 방법으로 진행하여 목적화합물인 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-Lys(Alloc)-Ala-Arg(Pbf)-Lys(Boc)-Glu(OAll)-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib- His(Trt)-Boc 을 얻었다.Proceed in the same manner as in step (3) of Synthesis 1) Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val- Phe-Glu (OtBu) -Lys (Alloc) -Ala-Arg (Pbf) -Lys (Boc) -Glu (OAll) -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp (OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-His (Trt) -Boc.
비교예 3: 고리화 폴리펩타이드의 제조Comparative Example 3: Preparation of Cyclic Polypeptide
단계 (1): 부분 탈보호 제조 (Alloc/OAll)Step (1): Partial Deprotection Preparation (Alloc / OAll)
용기에 테트라키스팔라듐 11.5g과 디클로로메탄 800㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 N-메틸아닐린 78.6㎖와 페니실란 38.5㎖을 투입하고 5분 동안 교반시켰다. 상기 합성 30)에서 제조한 폴리펩타이드(폴리 30mer 합성)에 디클로로메탄 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 만들어 놓은 반응액을 여과된 수지에 투입하고 실온에서 5시간 동안 교반한 후 여과하였다. 여과된 수지에 디클로로메탄 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 5회 실시하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 2회 실시하였다.11.5 g of tetrakispalladium and 800 ml of dichloromethane were added to the vessel, followed by stirring to completely dissolve it. 78.6 ml of N-methylaniline and 38.5 ml of penicsilane were added to the dissolved reaction solution, and the mixture was stirred for 5 minutes. 480 mL of dichloromethane was added to the polypeptide prepared in Synthesis 30) (poly 30mer synthesis), stirred for 2 minutes, and filtered. This process was carried out three times in total. The prepared reaction solution was added to the filtered resin, stirred at room temperature for 5 hours, and filtered. 480 ml of dichloromethane was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out a total of five times. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out twice.
단계 (2): 고리화 폴리펩타이드의 제조 (고리화 폴리 30mer 합성)Step (2): Preparation of Cyclic Polypeptide (cyclic Poly 30mer Synthesis)
용기에 HATU 56.6g과 디메틸포름아미드 480㎖을 투입하고 교반하여 완전히 용해시켰다. 상기 용해시킨 반응액에 N,N-디이소프로필에틸아민 51.8㎖을 투입하고 상온에서 5분 동안 교반하였다. 상기 단계 (1)에서 제조한 폴리펩타이드(부분 탈보호 30mer)에 만들어 놓은 반응액을 투입하고 상온에서 3시간 동안 교반한 후 여과하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 여과된 수지에 메탄올 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 여과된 수지에 디메틸포름아미드 480㎖을 투입하고 2분 동안 교반한 후 여과하였다. 이 과정을 총 3회 실시하였다. 상기 과정을 거쳐 목적화합물인 고리화 폴리펩타이드 Resin-Cys(Trt)-Thr(tBu)-Asn(Trt)-Met-Leu-Trp(Boc)-Gln(Trt)-Val-Phe-Glu(OtBu)-cyclo-[Lys-Ala-Arg(Pbf)-Lys(Boc)-Glu]-Asp(OtBu)-Leu-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Tyr(tBu)-Asp(OtBu)-Ser(tBu)-Thr(tBu)-Phe-Thr(tBu)-Gly-Gln(Trt)-Aib-His(Trt)-Boc 을 얻었다.56.6 g of HATU and 480 ml of dimethylformamide were added to the vessel, followed by stirring to dissolve completely. 51.8 mL of N, N-diisopropylethylamine was added to the dissolved reaction solution, and the mixture was stirred at room temperature for 5 minutes. The reaction solution prepared in the polypeptide (partial deprotection 30mer) prepared in step (1) was added thereto, stirred at room temperature for 3 hours, and filtered. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. Methanol 480ml was added to the filtered resin, stirred for 2 minutes, and filtered. This process was carried out three times in total. 480 ml of dimethylformamide was added to the filtered resin, followed by stirring for 2 minutes, followed by filtration. This process was carried out three times in total. Cyclic polypeptide Resin-Cys (Trt) -Thr (tBu) -Asn (Trt) -Met-Leu-Trp (Boc) -Gln (Trt) -Val-Phe-Glu (OtBu) -cyclo- [Lys-Ala-Arg (Pbf) -Lys (Boc) -Glu] -Asp (OtBu) -Leu-Tyr (tBu) -Lys (Boc) -Ser (tBu) -Tyr (tBu) -Asp ( OtBu) -Ser (tBu) -Thr (tBu) -Phe-Thr (tBu) -Gly-Gln (Trt) -Aib-His (Trt) -Boc was obtained.
비교예 4: NH 2-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu]-Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-H, TFA 의 제조 (보호기 및 레진 절단, cleavage)Comparative Example 4: NH 2 -Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu] -Asp-Leu-Tyr-Lys-Ser -Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-H, Preparation of TFA (protector and resin cleavage, cleavage)
실시예 4와 동일하게 제조하였으며 목적 화합물인 NH2-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo-[Lys-Ala-Arg-Lys-Glu]-Asp-Leu-Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-H, TFA 을 얻었다. Prepared in the same manner as in Example 4, and a target compound NH2-Cys-Thr-Asn-Met-Leu-Trp-Gln-Val-Phe-Glu-cyclo- [Lys-Ala-Arg-Lys-Glu] -Asp-Leu -Tyr-Lys-Ser-Tyr-Asp-Ser-Thr-Phe-Thr-Gly-Gln-Aib-His-H, TFA was obtained.
RemarkRemark Area (%)Area (%)
실시예Example 비교예Comparative example
Imp. (max)Imp. (max) 14.414.4 28.628.6
PurityPurity 61.661.6 18.018.0
YieldYield 15 %15% 2 %2 %
상기의 실시예에 따라 고리화된 15mer 중간체를 먼저 합성 후 마지막 30mer sequence까지 합성하여 crude한 생리활성 폴리펩타이드 화합물을 합성할 경우 상기 비교예에 따라 1~30mer sequence까지 linear 합성 후 마지막에 고리화 공정을 진행하여 crude한 생리활성 폴리펩타이드 화합물을 합성한 경우와 비교하여 순도에서 훨씬 향상된 결과를 보여주었다. 또한, max impurity를 비교할 때 절반 수준으로 감소한 것을 확인할 수 있었다. 뿐만 아니라, 정제 후 수율을 비교해 보면 본 발명의 제조방법을 통하여 제조할 경우 15%를 나타내어 비교예의 2%와 비교하여 약 7.5배의 수율 차이를 있음을 확인할 수 있었다. 따라서, 고순도의 Crude한 생리활성 폴리펩타이드 화합물의 제조가 가능하다면 Prep. HPLC 등에 의한 정제 공정 진행 시 생산성과 효율성에서 향상될 것이다. 특히, Prep HPLC에 의한 정제 시 Crude 화합물의 순도가 높을수록 정제 효율도 좋아지고 보다 순수한 목표 화합물을 얻을 수 있으므로 최종의 목표 화합물인 생리활성 폴리펩타이드 화합물을 고수율, 고순도로 얻을 수 있는 장점이 있다. In the case of synthesizing the crude physiologically active polypeptide compound by first synthesizing the cyclized 15mer intermediate according to the above embodiment and then synthesizing the final 30mer sequence, the cyclization process is performed after linear synthesis up to 1-30mer sequence according to the comparative example. In comparison with the synthesis of crude bioactive polypeptide compounds showed a much improved results in purity. Also, when comparing the max impurity, it was confirmed that the level was reduced by half. In addition, when comparing the yield after purification it was confirmed that the manufacturing process through the manufacturing method of the present invention shows a 15% yield difference of about 7.5 times compared to 2% of the comparative example. Therefore, if it is possible to prepare high purity Crude bioactive polypeptide compounds, Prep. It will be improved in productivity and efficiency during the purification process by HPLC. In particular, the higher the purity of the Crude compound when purified by Prep HPLC, the higher the purification efficiency and the more pure target compound can be obtained, there is an advantage that can obtain the final target bioactive polypeptide compound in high yield, high purity .

Claims (16)

  1. 하기 화학식 1의 펩타이드 중간체 화합물:Peptide intermediate compounds of formula
    [화학식 1][Formula 1]
    Figure PCTKR2019008935-appb-img-000006
    Figure PCTKR2019008935-appb-img-000006
    : R-Cyclo(-Glu-Lys-Arg-Ala-Lys)-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-X.: R-Cyclo (-Glu-Lys-Arg-Ala-Lys) -Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-X.
    여기서, R은 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되고;Wherein R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 Cycloalkyl, heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkylC 6-12 aryl, C 1-6 alkylC 6-12 aryloxycarbonyl and heteroaryl Selected from the group consisting of;
    X는 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되며;X is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
    상기 언급된 치환체들은 추가적으로 H, 할로젠, 시아노, 직쇄형 또는 분지형의 C 1-6알킬, 직쇄형 또는 분지형의 C 2-10알켄일, C 3-10사이클로알킬, 할로C 1-5알킬, 하이드록시C 1-6알킬, 아미노, 모노 또는 디C 1-6알킬아미노, 옥소, 하이드록시, C 1-6알콕시, C 6-12아릴설포닐 및 C 1-6알킬설포닐로 이루어진 군에서 선택된 1종 이상의 동일하거나 상이한 치환기로 치환될 수 있다. The aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
  2. 제1항에 있어서, The method of claim 1,
    상기 R은 H, 직쇄형 또는 분지형의 C 1-12알킬옥시, C 6-12아릴옥시, 및 C 1-6알킬C 6-12아릴옥시로 이루어진 군으로부터 선택되는 것인 펩타이드 중간체 화합물.Wherein R is selected from the group consisting of H, straight or branched C 1-12 alkyloxy, C 6-12 aryloxy, and C 1-6 alkylC 6-12 aryloxy.
  3. 제2항에 있어서, The method of claim 2,
    상기 R은 tert-부틸옥시 또는 9-플루오레닐메틸옥시인 펩타이드 중간체 화합물.R is a tert-butyloxy or 9-fluorenylmethyloxy peptide intermediate compound.
  4. 제1항에 있어서, The method of claim 1,
    상기 R이 H이고, X가 H인 펩타이드 중간체 화합물.The peptide intermediate compound wherein R is H and X is H.
  5. 하기 화학식 3의 레진 복합체 화합물:Resin complex compounds of formula
    [화학식 3][Formula 3]
    Figure PCTKR2019008935-appb-img-000007
    Figure PCTKR2019008935-appb-img-000007
    여기서, A 내지 D는 보호기이며;Wherein A to D are protecting groups;
    A 내지 D는 각각 독립적으로 트리페닐메틸(Trt), 터셔리부틸(tBu), t-부틸옥시카보닐(Boc) 및 2,2,4,6,7-펜타메틸디하이드로벤조퓨란-5-설포닐(Pbf)로 이루어진 군으로부터 선택되고,A to D are each independently triphenylmethyl (Trt), tertiary butyl (tBu), t-butyloxycarbonyl (Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5- Selected from the group consisting of sulfonyl (Pbf),
    R은 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되고;R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
    X'은 레진이며;X 'is a resin;
    상기 언급된 치환체들은 추가적으로 H, 할로젠, 시아노, 직쇄형 또는 분지형의 C 1-6알킬, 직쇄형 또는 분지형의 C 2-10알켄일, C 3-10사이클로알킬, 할로C 1-5알킬, 하이드록시C 1-6알킬, 아미노, 모노 또는 디C 1-6알킬아미노, 옥소, 하이드록시, C 1-6알콕시, C 6-12아릴설포닐 및 C 1-6알킬설포닐로 이루어진 군에서 선택된 1종 이상의 동일하거나 상이한 치환기로 치환될 수 있다.The aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
  6. 제5항에 있어서, The method of claim 5,
    상기 레진은 폴리스티렌 (PS) 계열 레진 또는 폴리스티렌-폴리에틸렌 글리콜 공중합체 (PS-PEG copolymer) 계열의 레진인 레진 복합체 화합물.The resin is a resin composite compound of a polystyrene (PS) resin or a polystyrene-polyethylene glycol copolymer (PS-PEG copolymer) resin.
  7. 제6항에 있어서, The method of claim 6,
    상기 레진은 아미드 링크 MBHA인 레진 복합체 화합물.The resin is a resin complex compound is an amide link MBHA.
  8. (1) 극성 비양성자성 용매 중에서 레진을 팽윤시키는 단계;(1) swelling the resin in a polar aprotic solvent;
    (2) 극성 비양성자성 용매 중에서 피페리딘 용액을 사용하여 보호기를 탈보호화시켜 탈보호화된 레진을 제조하는 단계; (2) deprotecting the protecting group using a piperidine solution in a polar aprotic solvent to prepare a deprotected resin;
    (3) 극성 비양성자성 용매 중에 보호화된 아미노산, 1-히드록시-1H-벤조트리아졸 및 1,3-디이소프로필카보디이미드를 가하여 보호화된 아미노산을 활성화시키는 단계;(3) adding protected amino acids, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide in a polar aprotic solvent to activate the protected amino acids;
    (4) 반응기 내 탈보호화된 레진에 활성화된 보호화된 아미노산 용액을 가하여 커플링하는 단계;(4) adding and coupling an activated protected amino acid solution to the deprotected resin in the reactor;
    (5) 펩타이드가 형성될 때까지 단계 (2)-(4)를 반복하는 단계;(5) repeating steps (2)-(4) until the peptide is formed;
    (6) 합성된 펩타이드를 용매 하에서 테트라키스팔라듐, N-메틸아닐린 및 페니실란과 반응시켜 부분 탈보호화된 레진을 제조하는 단계; 및(6) reacting the synthesized peptide with tetrakispalladium, N-methylaniline and penicsilane in a solvent to produce partially deprotected resin; And
    (7) 극성 비양성자성 용매 중에 합성된 펩타이드와 커플링 시약을 가하여 고리화하는 단계(7) cyclization by adding the coupling peptide and the synthesized peptide in a polar aprotic solvent
    를 포함하는, 하기 화학식 3의 레진 복합체 화합물의 제조방법:A method of preparing a resin complex compound of Formula 3, comprising:
    [화학식 3][Formula 3]
    Figure PCTKR2019008935-appb-img-000008
    Figure PCTKR2019008935-appb-img-000008
    여기서, A 내지 D는 보호기이며;Wherein A to D are protecting groups;
    A 내지 D는 각각 독립적으로 트리페닐메틸(Trt), 터셔리부틸(tBu), t-부틸옥시카보닐(Boc) 및 2,2,4,6,7-펜타메틸디하이드로벤조퓨란-5-설포닐(Pbf)로 이루어진 군으로부터 선택되고,A to D are each independently triphenylmethyl (Trt), tertiary butyl (tBu), t-butyloxycarbonyl (Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5- Selected from the group consisting of sulfonyl (Pbf),
    R은 H, 직쇄형 또는 분지형의 C 1-12알킬, 직쇄형 또는 분지형의 C 1-12알킬옥시카보닐, 직쇄형 또는 분지형의 C 2-12알켄일, C 3-10사이클로알킬, 헤테로사이클로알킬, C 6-12아릴, C 6-12아릴옥시카보닐, C 1-6알킬C 6-12아릴, C 1-6알킬C 6-12아릴옥시카보닐 및 헤테로아릴로 이루어진 군으로부터 선택되고;R is H, straight or branched C 1-12 alkyl, straight or branched C 1-12 alkyloxycarbonyl, straight or branched C 2-12 alkenyl, C 3-10 cycloalkyl , Heterocycloalkyl, C 6-12 aryl, C 6-12 aryloxycarbonyl, C 1-6 alkyl C 6-12 aryl, C 1-6 alkyl C 6-12 aryloxycarbonyl and heteroaryl Is selected from;
    X'은 레진이며;X 'is a resin;
    상기 언급된 치환체들은 추가적으로 H, 할로젠, 시아노, 직쇄형 또는 분지형의 C 1-6알킬, 직쇄형 또는 분지형의 C 2-10알켄일, C 3-10사이클로알킬, 할로C 1-5알킬, 하이드록시C 1-6알킬, 아미노, 모노 또는 디C 1-6알킬아미노, 옥소, 하이드록시, C 1-6알콕시, C 6-12아릴설포닐 및 C 1-6알킬설포닐로 이루어진 군에서 선택된 1종 이상의 동일하거나 상이한 치환기로 치환될 수 있다.The aforementioned substituents may additionally be H, halogen, cyano, straight or branched C 1-6 alkyl, straight or branched C 2-10 alkenyl, C 3-10 cycloalkyl, haloC 1- 5 alkyl, hydroxyC 1-6 alkyl, amino, mono or diC 1-6 alkylamino, oxo, hydroxy, C 1-6 alkoxy, C 6-12 arylsulfonyl and C 1-6 alkylsulfonyl It may be substituted with one or more same or different substituents selected from the group consisting of.
  9. 제8항에 있어서, The method of claim 8,
    상기 극성 비양성자성 용매는 디메틸포름아미드, 디메틸아세트아미드 및 이들의 혼합물로 이루어진 군으로부터 선택되는 것을 특징으로 하는 레진 복합체 화합물의 제조방법.Wherein said polar aprotic solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, and mixtures thereof.
  10. 제8항에 있어서, The method of claim 8,
    상기 단계 (6)에서 용매는 디클로로메탄, 클로로포름 및 이들의 혼합물로 이루어진 군으로부터 선택되는 것을 특징으로 하는 레진 복합체 화합물의 제조방법.The solvent in step (6) is a method for producing a resin composite compound, characterized in that selected from the group consisting of dichloromethane, chloroform and mixtures thereof.
  11. 제8항에 있어서, The method of claim 8,
    상기 단계 (7)에서 커플링 시약은 1-히드록시-1H-벤조트리아졸/1,3-디이소프로필카보디이미드 또는 HATU/N,N-디이소프로필에틸아민으로부터 선택되는 것을 특징으로 하는 레진 복합체 화합물의 제조방법.The coupling reagent in step (7) is selected from 1-hydroxy-1H-benzotriazole / 1,3-diisopropylcarbodiimide or HATU / N, N-diisopropylethylamine Method for preparing a resin complex compound.
  12. (1) 제8항의 방법으로 제조한 고리화된 펩타이드 화합물을 극성 비양성자성 용매 중에서 피페리딘 용액을 사용하여 보호기를 탈보호화시켜 탈보호화된 레진을 제조하는 단계; (1) deprotecting a cyclized peptide compound prepared by the method of claim 8 using a piperidine solution in a polar aprotic solvent to prepare a deprotected resin;
    (2) 극성 비양성자성 용매 중에 보호화된 아미노산, 1-히드록시-1H-벤조트리아졸 및 1,3-디이소프로필카보디이미드를 가하여 보호화된 아미노산을 활성화시키는 단계;(2) adding protected amino acid, 1-hydroxy-1H-benzotriazole and 1,3-diisopropylcarbodiimide in a polar aprotic solvent to activate the protected amino acid;
    (3) 반응기 내 탈보호화된 레진에 활성화된 보호화된 아미노산 용액을 가하여 커플링하는 단계;(3) adding and coupling an activated protected amino acid solution to the deprotected resin in the reactor;
    (4) 펩타이드가 형성될 때까지 단계 (1)-(3)을 반복하는 단계;(4) repeating steps (1)-(3) until the peptide is formed;
    (5) 절단 칵테일을 사용하여 보호화된 레진을 탈보호화시키는 동시에 레진으로부터 원하는 펩타이드를 절단하는 단계; 및(5) cleaving the desired peptide from the resin while simultaneously deprotecting the protected resin using the cleavage cocktail; And
    (6) 레진으로부터 절단 혼합물을 여과하는 단계(6) filtering the cleavage mixture from the resin
    를 포함하는, 하기 화학식 2의 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염의 제조방법. A method of preparing a physiologically active polypeptide of the formula (2) and a pharmaceutically acceptable salt comprising a.
    [화학식 2][Formula 2]
    Figure PCTKR2019008935-appb-img-000009
    Figure PCTKR2019008935-appb-img-000009
    : H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Ser-Lys-Tyr-Leu-Asp- Cyclo(-Glu-Lys-Arg-Ala-Lys)-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-NH 2 : H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Ser-Lys-Tyr-Leu-Asp- Cyclo (-Glu-Lys-Arg-Ala- Lys) -Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys-NH 2
  13. 제12항에 있어서, The method of claim 12,
    상기 극성 비양성자성 용매는 디메틸포름아미드, 디메틸아세트아미드 및 이들의 혼합물로 이루어진 군으로부터 선택되는 것을 특징으로 하는 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염의 제조방법. And wherein said polar aprotic solvent is selected from the group consisting of dimethylformamide, dimethylacetamide, and mixtures thereof.
  14. 제12항에 있어서, The method of claim 12,
    상기 절단 칵테일은 트리플루오로아세트산 (TFA), 1종 이상의 스캐빈저 및 디클로로메탄의 용액을 포함하하는 것을 특징으로 하는 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염의 제조방법. Wherein said cleavage cocktail comprises a solution of trifluoroacetic acid (TFA), at least one scavenger, and dichloromethane.
  15. 제14항에 있어서, 상기 스캐빈저가 트리이소프로필실란 (TIPS), 트리에틸실란 (TES), 페놀, 아니솔, 티오아니솔, 물, 에탄디티올 (EDT), 1-도데칸티올, 디티오트레이톨 (DTT) 및 인돌로 이루어진 군으로부터 선택되는 것을 특징으로 하는 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염의 제조방법. The method of claim 14, wherein the scavenger is triisopropylsilane (TIPS), triethylsilane (TES), phenol, anisole, thioanisole, water, ethanedithiol (EDT), 1-dodecanethiol, dithi A process for preparing a physiologically active polypeptide and a pharmaceutically acceptable salt, characterized in that it is selected from the group consisting of othritol (DTT) and indole.
  16. 제12항 내지 제15항 중 어느 한 항에 있어서, The method according to any one of claims 12 to 15,
    상기 약학적으로 허용가능한 염은 트리플루오로아세트산인 것을 특징으로 하는 생리활성 폴리펩타이드 및 약학적으로 허용가능한 염의 제조방법.The pharmaceutically acceptable salt is a trifluoroacetic acid, characterized in that the physiologically active polypeptide and a method for producing a pharmaceutically acceptable salt.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7928058B2 (en) * 2006-02-22 2011-04-19 Merck Sharp & Dohme Corp. Pharmaceutical composition comprising oxyntomodulin derivatives and a method for reducing body weight using the composition
KR20140018462A (en) * 2012-07-25 2014-02-13 한미약품 주식회사 A composition for treating hyperlipidemia comprising oxyntomodulin analog
KR20140058104A (en) * 2012-11-06 2014-05-14 한미약품 주식회사 A composition for treating diabetes or diabesity comprising oxyntomodulin analog
KR20140113696A (en) * 2011-12-23 2014-09-24 입센 메뉴팩츄링 아일랜드 리미티드 Process for the synthesis of therapeutic peptides
KR20170003466A (en) * 2015-06-30 2017-01-09 한미약품 주식회사 Novel glucagon derivative and a composition comprising a long acting conjugate of the same

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US7928058B2 (en) * 2006-02-22 2011-04-19 Merck Sharp & Dohme Corp. Pharmaceutical composition comprising oxyntomodulin derivatives and a method for reducing body weight using the composition
KR20140113696A (en) * 2011-12-23 2014-09-24 입센 메뉴팩츄링 아일랜드 리미티드 Process for the synthesis of therapeutic peptides
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