TWI282340B - Solid phase peptide synthesis - Google Patents

Description

1282340 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method for preparing a peptide derivative of the following formula:

Among them, Tos means p-toluenesulfonyl. [Prior Art] The method of the present invention is based on solid phase synthesis. During solid phase synthesis, the starting material is bonded to the inert solid support while the amino acid is combined (i.e., coupled) to the peptide of any desired sequence. The reactants are added to the solution because the starting product is bonded to the solid, so any product derived from the starting material also remains bonded. Once the desired sequence is attached to the support, the peptide is detached from the support (i.e., cleavage). The peptide derivatives prepared according to the invention are suitable for the quantitative determination of certain proteolytic enzymes of the class EC 3·4·4 and are particularly suitable for the quantitative determination of thrombin (^. For the International Biochemical Union'' Enzyme Commission (Enzyme c〇) mmiUee wide shrink

Liquid phase synthesis by acid derivatives.

104995.doc 1282340 Efforts to study the purity and purification required for the respective peptides are still not satisfactory [invention] ^ / /, the object of the present invention is to provide a peptide derived from the production of the peptide of formula 1 in good yield and high optical purity. A more economical method of things. This object is achieved by the method of the invention as claimed in claim 1. The method comprises: human a) continuously coupling an amino acid arginine, a proline and a glycine on a solid support in the presence of a coupling agent/additive system, b) a sulfonium sulfonate glycine moiety Να_amine, c) cleavage of the toluene sulfonated peptide or its amine side chain protected derivative from the solid support, d) peptide intermediate of the formula:

Or an amine side chain protected derivative thereof and an aniline of the formula:

R-NH2III/, R represents a p-aminophenyl group and one of the amine groups is protected with an amine protecting group in the presence of an even 5 agent/additive system. [Embodiment] 104995.doc 1282340 The meanings of the abbreviations used in the description of the invention and the scope of the patent application are summarized in the following table: Fmoc 9 - mercapto decyloxy-Weilyl- Boc tert-butoxycarbonyl- Tos 4-曱Benzenesulfonyl-DIEA Diisopropylethylamine NMP N-decylpyrrolidone DCM Dichlorodecane TFA Trifluoroacetic acid DMF Ν, Ν1-dimethylformamide HBTU hexafluoro-phosphate 0-benzoquinone Azathioprine, anthracene, Ν', Ν'-tetramethyl-urea rust HOBt 1-hydroxybenzotriazole HOOBt 3,4-diaza-3-yl-4-yl-1,2,3-benzoquinone Di-DEPBT 3-(diethoxyphosphoniumoxy)-1,2,3-benzotriazine-4(3Η)-one PyBOP hexafluoro-acid (benzotriazole-1-yloxy) Acetidone scale CTC 2 - gas diphenyl sulfhydryl gas DCC Ν, Ν '- dicyclohexylcarbodiimide TBTU tetrafluoroborate 0-(benzotriazol-1-yl)-Ν, Ν, Ν ',Ν'-tetradecylurea rust HOAt 1-Loliki-7-azabenzaldehyde dip. Pbf 5-sulfonyl-2,2,4,6,7-pentamethylenebenzofuran Pmc 6 -sulfonyl-2,2,5,7,8-pentamethylchromate Et3N triethylamine 104995.doc 1282340 - should be improved to understand the amino acid arginine and proline L_ configuration or a - D, 'and a variety of evil as a racemate or a mixture of isomers. The amino acid such as hydrazine is preferably used in its oxime configuration. "Continuous coupling in step a) of the invention comprises the first step of attaching a preferably protected arginine to a solid support. The alpha-amine group of arginine may be known to those skilled in the art. It is known to protect the common amine protecting group. Fm〇c is the preferred cardioprotective group of arginine. • The side chain of the arginine molecule (ie, the oxime moiety) is generally familiar with the technology. It is known that the arginine side chain protecting group is protected. The car is good for the side chain. The mouth group is pmc or Pbf, and more preferably Pbf. In principle, it is known that each solid phase carrier suitable for solid phase peptide synthesis can be used in the synthesis of the present invention, as described in the following literature on peptides. Chemist7 and

Bl〇l〇gy, N. Sewald, H.-D. Jakubke, Wiley-VCH Verlag

GmbH, Weinheim, 2002 and Fmoc-Solid Phase Peptide Synthesis-A practical approach, W. C. Chan, P. D. White, Oxford University Press inc. New York, 2000. It has been found that 2-gastriphenylhydrazine-based gas-polystyrene resin (Ctc resin) is most suitable as a solid phase carrier for the purpose of peptide synthesis of the present invention. CTC resins are commercially available, for example, from Merck Bioscience. The protected arginine is preferably dissolved in an inert solvent such as, for example, dichloromethane. There are usually tertiary amines such as EtsN, DIEA or symmetric trimethylpyridine, preferably DIE A or symmetric trimethylpyridine. Attachment to a solid support is usually carried out at room temperature. 104995.doc 1282340 The treatment of the loaded resin follows techniques known to those skilled in the art and involves washing the resin with an organic solvent, filtering, and finally drying at an appropriate temperature. In a preferred embodiment of this first step, a CTC tree loaded with Fmoc-Arg(Pbf)-OH is prepared. In a subsequent step, it is coupled to a protected proline, followed by coupling with a protected glycine. The alpha-amino protecting group of arginine should be in a 5-20% solution of a suitable solvent such as DMF or NMP, preferably NMP, by means of, for example, morphine, DBU or brigade bit before the coupling can proceed. Jia is a bit of a secondary amine that is conveniently protected. The alpha-amine groups of the proline and glycine acids can be protected by conventional amine protecting groups known to those skilled in the art. For valine and glycine, Fmoc is a preferred a-amino protecting group. The proline may be selected from the form of a reactive derivative of PG-Pro-OPfp, PG-Pro-OSu and PG-Pro-OBt, or an inactive derivative in the form of PG-Pro-OH, wherein PG means an amine Base protection base. Proline is preferably applied in the form of Fmoc-(L)-Pro-〇H. Glycine can be used as an active derivative in the form of PG-Gly-OPfp and PG-Gly-OSu or as an inactive derivative in the form of PG-Gly-OH, wherein PG means an amine protecting group. Glycine is preferably used in the form of Fmoc-Gly-OH. According to the present invention, the amino acid coupling uses a coupling agent/addition agent system selected from the group consisting of DCC/HOBt, HBTU/HOBt, TBTU/HOBt, HATU/HOAt, DEPBT/H〇〇Bt, PyBoP/Cl-H〇Bt To reach. 104995.doc 1282340 - The preferred coupler/additive system is DEPBT/HOOBt or HBTU/HOBt, whereby HBTU/H〇Bt is preferred. Each coupling is generally carried out in a suitable solvent such as a solvent in the presence of a tertiary amine such as yoghurt, DIEA or symmetrical trimethylpyridine, preferably DIEA or symmetrical trimethylsulfonium. The coupling reaction is ideally from 〇 t to 4 〇. (3) carried out under temperature agitation. Deprotection of proline after the coupling with glycine and final deprotection of glycine can be carried out in a 5-20% solution of a suitable solvent such as DMF or NMP, preferably NMP. The secondary amine which is preferably bitten by means of, for example, TBU or DBU, is conveniently achieved. In a preferred embodiment of the coupling reaction, a load of H2N_G1" (L)-Pro-(D/L) is prepared. CTC resin of Arg_(Pbf)-〇H. After the step of the method of the present invention, the oxime-α-amine group of the toluene sulfonated glycine moiety is usually in, for example, 玢川, mEA or symmetrical trimethylpyridine. It is preferably carried out in the presence of a tertiary amine of DIEA with toluenesulfonyl chloride. _ Toluene sulfonation is usually carried out in the presence of an inert solvent such as dichloromethane at a temperature of from 〇C to 40C. Cleavage of the vector can be accomplished by those skilled in the art. / The desired peptide T〇s-Gly-(L)-Pr〇-(L)_Arg(Pbf)-〇H is preferably by thinning The acidic solution is preferably cleaved from the CTC resin by treatment with a solution of M-5% trifluoroacetic acid in dioxane. The peptide obtained can preferably be borrowed by methods known in the art. Liquid chromatography purification technique 104995.doc 1282340 _ further purified according to step d), the acylated peptide toluenesulfonamide derivative with an aniline of the formula:

R-NH2 III wherein R means p-aminophenyl, wherein one of the amine groups is protected by an alpha-amino protecting group and is coupled in the presence of a coupling/additive system. The coupling is in the presence of a coupling/additive system selected from the group consisting of DCC/HOBt, HBTU/HOBt, TBTU/HOBt, HATU/HOAt, PyBOP/Cl-HOBt, DEPBT/HOOBt, preferably DEPBT/HOOBt, such as Et3N, DIEA Or in the presence of a symmetric trimethylpyridine, preferably a tertiary amine of DIEA. The reaction is preferably carried out under stirring at a temperature of from C ° C to 40 ° C in a suitable solvent such as DMF. The treatment of the reaction mixture is carried out in accordance with the common knowledge of those skilled in the art and involves extraction with a dilute acid such as dilute hydrochloric acid. The peptide obtained can be further purified by liquid phase chromatography purification techniques preferably by methods known in the art. p The desired peptide isomer of up to 85% excellent yield and up to 96% optical purity can be obtained by the method of the present invention. Example 1

Fmoc-(L)-Arg(Pbf>OH attached to CTC resin 500 g (0.77 mol) Fmoc-(L)-Arg(Pbf) (Merck Biosciences Novobiochem) was dissolved in 5.5 1 dichloromethane and 812 ml (4.77 mol) ) in a stirred solution of DIEA. Add force σ 1 kg CTC resin (Merck Biosciences GmbH, 100-200 mesh, 1% DVB, load ·························· The mixture was subjected to 104995.doc 12.82340 month f·3 h at room temperature, wherein the mixture was stirred after 1 h and 2 respectively. After 3 h, the mixture was cooled to 5_1 (rc and 3 〇〇ml tau alcohol was added). The suspension was allowed to stand at this temperature for ih. The mixture was then transferred on a suction device. The resin was then suspended in a solution of dichloromethane/methanol/DIEA (8:15:5) and stirred. 5 min and allowed to stand for 30 min. After filtration, the resin was washed 4 times with 5] DMF, 4 times with 2.5 1 isopropyl alcohol and washed with 2. 5 1 isohexane.

Times. The resin should be filtered to maintain wetting. The resin was then dried in a vacuum oven at 30 ° C for 40 h. Analytical load by HPLC: 0.405 mmol/g Fmoc-Arg(Pbf)-CTC HPLC method: column: Keystone Beta Basic C18; mobile phase A: H20+0·1% TFA; mobile phase B: acetonitrile + 0·075% TFA; T = 3 0 C ; t = 22 min; Rt = 12.2 min 〇 Example 2:

Tos-Gly-(L)-Pro-(L)-Arg(Pbf)-OH

1) 5% piperidine in 1 coffee P 2) Fmo^Pro-OH.HBTU.HOBt.DlEA 3) 5% piperidine in HMF 4) FmooOf/.OH, HBTU, HOBt.DlEA 5) HHP 6) Tos -CI, DIEA 7) TFA·solution

A peptide reaction vessel was fed with 5 g of Fmoc-(L)-Arg(Pbf)-CTC-resin (negative 104995.doc 12 1282340 - ··405 mmol/g; 2-075 mmol). 87.5 ml of dioxane was added. When the far resin has been expanded in methylene chloride for at least 30 min, the solvent is changed to NMP. Next, washing with 87. 5 ml of the resin was completed (3 times). Deblocking was carried out in a solution of 5% piperidine in NMP over 30 min. The resin was then washed 6 times with 62.5 ml of NMP. The coupling procedure was prepared by preparing 1.05 g (3_n mm〇l) Fm〇c_(L)_pr〇_〇H, 0,477 g (3,ll mm〇l)H〇Bt and 1.09 ml (6.22 mmol) DIEA at 8.75 φ ml The solution in NMP was added after 1. 5 g of 1.18 g (3·11 mm〇l) HBTU in 7.5 mi NMP. The solution was added to the resin after preactivation of i 〇 min and the suspension was carefully stirred at 3 ° C for 2 h. After the coupling, it was washed thoroughly with NMP. Amino blocking was carried out in a solution of 5% piperidine in NMP over 30 min. The resin was then thoroughly washed with NMP. This coupling procedure was carried out by preparing a solution of 925·925 g (3·11 mmol) of Fmoc-Gly-OH. After 5 min, 0.477 g (3.11 mmol) of HOBt and 1·〇9 ml (622 • mmol) of 7·5 NMP were added. The solution was added to the resin after 10 min preactivation and the suspension was carefully stirred at 30 °C for 2 h. After the coupling, it was washed thoroughly with NMP. The final partitioning of the amine group was again carried out in a solution of 5% piperidine in NMP over 30 min, followed by extensive washing (3 times) with NMP and dichloromethane. The resin was suspended in 75 ml of dichloromethane and 〇475 g (2 49 nrnol) 4-nonylbenzenesulfonium chloride (Tos-Cl) and 0.43 ml (2.49 mm 〇1) mEA were added. The final cleavage was carried out in DCM with 1% TFA solution. The filtrate was diluted with toluene and evaporated in vacuo. I04995.doc 13 1282340 Mobile phase A: Η2Ο + 0·1% TFA, mobile phase B: acetonitrile + 0.1% TFA, T=30°C 5 t = 34 min5 Rt=16.11 min5 A%: 91.13% 5 HPLC, method 2: Column: Chirobiotic T; 10 m, 250 x 4.6 mm; isocratic mobile phase: ((acetonitrile: MeOH; 1:4) + 0.2% Et3N + 0.2% AcOH), T = 30 〇 C, t = 30 min, Rt = 4.53 min, A%: 95.4%, NMR: 4 and 13C. ESI-MS: MH+ 925.2, MNa+ 947.2, [M-H]-923.2.

104995.doc -15 -

Claims (1)

Patent Application No. T9 89$®fV35422 Please replace the patent scope (February 2nd) Patent application scope: 1. A method for preparing a peptide derivative of the following formula or a salt thereof, Wherein Tos means p-toluenesulfonyl, the method comprises: a) continuously coupling amino acid arginine, lysine and glycine on a solid support in the presence of a coupling agent/additive system, wherein The solid support is 2-chlorotrityl chloride-polystyrene, b) the oxime-α-amine group of the benzene sulfonate glycine, c) the toluene sulfonate cleavage from the solid support a peptide or an amine side chain protected derivative thereof, d) a peptide intermediate of the formula: Or an amine side chain protected derivative thereof and an aniline of the formula: III R-NH 104995-960205.doc 1282340 I wherein R means an aminophenyl group and one of the amine groups is protected by an amine protecting group , react in the presence of a coupling agent/additive system. 2. The method of claim 1, wherein the arginine is a side chain protected by a Pbf or pmc protecting group. 3. The method of claim 1, characterized in that the proline is selected from the group consisting of PG-Pro-Pfp, PG_Pro-OSu and PG-Pro_〇Bt, or in the form of PG-Pro-OH Inactive derivative applications wherein PG means an amine protecting group. ® 4. The method of claim 3, characterized in that the proline is applied in the form of Fmoc-(L)-Pro_OH. 5. The method of claim 4, wherein the Fmoc protecting group is removed by treatment with piperidine. 6. The method of claim 1, characterized in that the glycine acid is selected from the group consisting of PG-Gly-OPfp, PG-Gly-OSu and PG-Gly-OBt, or in the form of PG-Gly-OH Inactive derivative applications wherein PG means an amine protecting group. The method of claim 6, characterized in that the glycine is applied in the form of Fmoc-Gly-OH. 8. The method of claim 7, wherein the Fmoc protecting group is removed by treatment with piperidine. 9. The method of claim 1 wherein the coupling agent/additive system for the continuous coupling of the amino acid in step a) is selected from the group consisting of DCC/HOBt, HBTU/HOBt, TBTU/HOBt, HATU/HOAt, DEPBT/ HOOBt, PyBoP/Cl-HOBt. 104995-960205.doc i 1282340 -- The method of claim 9, characterized in that the coupling agent/additive system is HBTU/HOBt. 11_ The method of claim 1, wherein the coupling agent/additive system for reacting the peptide intermediate of formula II with the aniline of formula III is selected from the group consisting of DCC/HOBt, HATU/HOAt, HBTU/HOBt, TBTU/HOBt, PyBOP/Cl-HOBt and DEPBT/HOOBt. 12. The method of claim 11, wherein the coupling agent/additive system is DEPBT/HOOB, and the method of claim 1, wherein the benzene sulfonate is 4-toluene sulfonium chloride. get on. The method of claim 1, wherein the cleavage from the solid phase support is carried out with trifluoroacetic acid. 15. The method of claim 1, wherein the reaction with aniline of formula III in step d) is carried out using Tos_Gly-Pro-Arg(Pbf)-OH as the amine side chain of the peptide intermediate of formula II. Derivatives are carried out. 104995-960205.doc