RU2340626C1 - Method of obtaining dodecapeptide and tripeptide for its realisation - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of obtaining dodecapeptide of the formula I: H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH and tripeptide of the formula II: X-Asp(Y)-His-Leu-OH is the intermediate compound in its synthesis. Solid-phase synthesis of dodecapeptide I is realised by sequential growth of the peptide chain, beginning with the C-end dipeptidilpolymertill the obtaining of C-end nonapeptidilpolimer, which is condensed with the protected N-end tripeptide of the formula II: X-Asp (Y)-His-Leu-OH where X, Y are protected groups and the obtained dodecapeptidilpolymer is processed with an unblocking agent for removing the protective groups and the polymeric matrix and in 1 stage the end product is given out by means of HELC.

EFFECT: increasing the output of the end product and simplification of the process.

3 cl, 2 ex

Description

The invention relates to the field of physiologically active peptides, specifically to a method for producing a dodecapeptide of formula I:

H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH (I), as well as to the tripeptide Boc-As (OBu ^t ) -His-Leu-OH of the formula ( II), which is an intermediate in its synthesis.

The dodecapeptide of formula I has the ability to inhibit the migration of THP-1 promonocytic cells and monocytes isolated from human blood under cell culture conditions, as well as the migration of monocytes and granulocytes to foci of inflammation in animals and can be used as a means to prevent the development of acute coronary conditions and prevent restenoses after angioplasty and stenting, as well as in acute inflammatory processes and exacerbation of various chronic diseases, in particular with gout and rheumatism idnom arthritis [1].

A known method for producing a dodecapapeptide of formula I by the solid-state method by stepwise peptide chain extension, starting from the C-terminal amino acid covalently linked to the polymer matrix, using the corresponding Fmoc amino acids in the presence of a condensing agent, followed by the release of the final dodecapapeptidyl polymer in 2 stages: treatment with piperidine solution and then trifluoroacetic acid, followed by purification of the obtained product using high performance liquid chromatography (HPLC) [2]. In this case, a dodecapeptide of formula I is obtained with a yield of 46% calculated on the starting amino acid attached to the polymer (see Figure 1, prototype).

The disadvantages of this method are the low yield of the target product and the multi-stage process. The disadvantage is the use of expensive and inaccessible derivatives of amino acids at the stages of peptide chain extension, starting from the C-terminal nonapeptyl polymer (V).

The above disadvantages make the known method unsuitable for large-scale synthesis of the dodecapapeptide of formula I.

The aim of the invention is to increase the yield of the target product and simplify the process.

This goal is achieved by the claimed method, according to which the solid-phase synthesis of the dodecapeptide of the formula:

carried out by sequentially increasing the peptide chain, starting from the C-terminal amino acid covalently linked to the polymer matrix, to obtain the C-terminal nonapeptidylpolymer (V), which is then condensed with a protected N-terminal tripeptide of the formula II:

where X, Y, are protective groups, after which the obtained dodecapeptidyl polymer is treated with a deblocking agent and all protective groups and the polymer matrix are cleaved in stage 1 and the final product is isolated by HPLC (see Appendix 2, the claimed method).

Synthesis intermediates used in the claimed method can be obtained by conventional peptide synthesis methods, in particular, by stepwise peptide chain growth in solution using activated esters of the corresponding protected amino acids [3].

List of abbreviations:

AcOH - acetic acid;

Boc is tert-butyloxycarbonyl;

Bu ^t is tert-butyl;

DIC - N, N ¹ -diizopropilkarbodiimid;

DCM - dichloromethane;

DMF — N, N-dimethylformamide;

DMSO - d6 - deuterated dimethyl sulfoxide;

Fmoc is 9-fluorenylmethoxycarbonyl;

HONSu - N-hydroxysuccinimide;

NOVT - 1-hydroxybenzotriazole;

HONp - para-nitrophenol;

NMP - N-methylpyrrolidone;

NMM is N-methylmorpholine;

Pip - piperidine;

TIBS - triisobutylsilane;

TFA - trifluoroacetic acid;

HPLC - high performance liquid chromatography;

TLC - thin layer chromatography;

"Complex F" - a complex of pentafluorophenol with dicyclohexylcarbodiimide in a ratio of 3: 1.

In the inventive method used derivatives of L-amino acids from Bachem (Switzerland), DIC, HONSu, HONp, NMP, TFA from Fluka (Switzerland). DMF was purified by distillation over ninhydrin and barium oxide. Analytical HPLC was carried out on a Gilson chromatograph (France), an Ultrasphere ODS column, 5 μm (4.6 × 250 mm) Beckman (USA) was used, buffer A — 0.05 M KH ₂ PO ₄ , pH 3.0, was used as eluents. buffer B - 70% acetonitrile in buffer A, elution with a concentration gradient from 0% to 60% of buffer B in buffer A for 40 min. A flow rate of 1 ml / min, detection at 220 nm. The individuality of the obtained compounds was confirmed by TLC on Kiselgel 60 chromatographic plates (Merck, Germany) in solvent systems: chloroform - methanol - acetic acid 9: 1: 0.5 (system 1), chloroform - methanol - 32% acetic acid 15: 4 : 1 (system 2), chloroform - methanol - 32% acetic acid 5: 3: 1 (system 3), chloroform - methanol - 32% acetic acid 60:45:20 (system 4), n. butanol - acetic acid - water 3: 1: 1 (system 5). The substances in the chromatograms showed chlorine - benzidine and ninhydrin. Melting points (uncorrected) were determined on a Boetius instrument (Germany). Amino acid analysis of peptides hydrolyzed 6 N. hydrochloric acid containing 2% phenol at 110 ° С for 24 h was carried out on a Biotronik LC 5001 instrument (Germany). ¹ H-NMR spectra were recorded on a WH-500 Broker 500 MHz spectrometer (Germany) in DMSO-d at 300 K, the concentration of peptides was 2-3 mg / ml. Chemical shifts were measured relative to tetramethylsilane. Mass spectra were recorded on a PC-Kompact MALDI instrument (Kratos, England).

Synthesis of Intermediates

Example 1. Boc-Asp (OBu ^t ) -His-Leu-OH (II):

9.52 g (20 mmol) of Boc-His (Boc) -ONp was dissolved in 100 ml of DMF, cooled to -10 ° C and 2.6 g (20 mmol) of leucine in 10 ml of 2N was added. NaOH and stirred at 20 ° C for 4 hours. The completion of the reaction was monitored by TLC in system 1. The reaction mixture was evaporated, the oily residue was dissolved in 200 ml of water and extracted with ether (3 × 50 ml). The aqueous phase was acidified with citric acid to pH 4, extracted with ethyl acetate (3 × 100 ml), the combined ethyl acetate extract was washed with water (3 × 50 ml), the solvent was removed in vacuo, the oily product was triturated with hexane. The result is 7.5 g (80%) of compound III. R _f 0.48 (1), 0.83 (2). The resulting material was crystallized from ether-hexane in the form of the corresponding dicyclohexylammonium salt. T. pl. 118-122 ° C.

To remove the Boc-protection, 6.0 g (12.8 mmol) of compound (III) are dissolved in 30 ml of TFA and incubated for 1 h at 20 ° C. Trifluoroacetic acid was evaporated, and H-His-Leu-OH trifluoroacetate was precipitated with ether. The result is 6.0 g (95%) of compound IV. R _f 0.27 (3), 0.32 (4), 0.13 (5).

6.0 g (12.2 mmol) of H-His-Leu-OH · TFA (IV) was dissolved in 100 ml of DMF, 2.7 ml (24.4 mmol) of NMM and 4.71 g (12.2 mmol) of Boc-Asp (OBu ^t ) - were added to the resulting solution. ONSu. The reaction mixture was kept for 18 hours, the end of the reaction was monitored by TLC in system 2. After the reaction was completed, the solution was evaporated, the residue was dissolved in 250 ml of a mixture of ethyl acetate and n-butanol (4: 1), 1 equivalent (12.2 mmol) of hydrochloric acid was added, washed water (3 × 70 ml) and evaporated. 100 ml of ether was added to the residue, the precipitate formed was filtered off, washed with ether (3 × 50 ml) and dried. The result is 5.4 g (82%) of compound II. R _f 0.45 (2), 0.75 (3), 0.82 (4). Mp 145-147 ° C. Signals (ppm) present in the ¹ H NMR spectrum:

1. Aspartic acid - 7.11 (NH ₂ , 2H), 4.26 (α-CH, 1H), 2.60, 2.38 (β-CH ₂ , 2H)

2. Histidine - 8.04 (NH, 1H), 4.59 (α-CH, 1H), 3.12, 2.96 (β-CH ₂ , 2H), 8.96 (C ₂ H, 1H, imidazole), 7.31 (C ₄ H, 1H imidazole)

3. Leucine - 8.16 (NH, 1H), 4.21 (α-CH, 1H), 1.53 (β-CH ₂ , 2H), 1.62 (γ-CH, 1H), 0.88, 0.83 (δ ', δ "-CH ₃ , 6H), as well as 1.37 (Boc, Bu ^t , 18H)

Example 2

2a. H-Asp (OBu ^t ) -Lys (Boc) -Gln (Trt) -Thr (Bu ^t ) -Gln (Trt) -Thr (Bu ^t ) -Pro-Lys (Boc) -Thr (Bu ^t ) -polymer ( V)

For solid-phase synthesis, a styrene copolymer with 1% divinylbenzene with a hydroxymethyl phenoxymethyl anchor group containing 0.67 mmol / g Fmoc-Thr (Bu ^t ) with a particle size of 200-400 mesh from Bachem (Switzerland) is used. The synthesis of the nonapeptidylpolymer is carried out starting from 0.37 g (0.25 mmol) of the Fmoc-Thr (Bu ^t ) -polymer in the automatic mode on the Applied Biosystems 431 A peptide synthesizer according to the standard program for a single condensation of Fmoc-amino acids.

The following protections are used to block the functional groups of the amino acid side chains: tert-butyl for the carboxyl groups of aspartic acid and the hydroxyl function of threonine; tert-butyloxycarbonyl (Boc) - protection for the ε-amino group of lysine; trityl (Trt) - a group for the carboxamide function of glutamine.

Solid Phase Synthesis Protocol No. Operation Reagent Time of processing one Flushing 5 × NMP 3 min 2 The release of α-amino groups 20% Pip / NMP 10 min 3 Flushing 5 × NMP 3 min Activation 1 mmol Fmoc amino acids + 1 mmol HOBt + 1 mmol DIC in NMP 20 minutes 6 Condensation 1 mmol activated derivative of Fmoc amino acids in NMP 90 min 7 Flushing 5 × NMP 3 min

To assess the quality of the intermediate V - C-terminal nonapeptidylpolymer - release and cleavage of the corresponding nonapeptide from the polymer carrier is carried out using a sample (50 mg) of intermediate V. Sample V is treated with 5 ml of a TFA solution containing 0.1 ml of deionized water and 0.1 ml of triisobutylsilane for 1 h, the polymer is filtered off, the filtrate is evaporated, ether is added to the residue and the precipitate is filtered off, washed with dichloromethane (3 × 1 ml), ether (3 times 1 ml), dried in a vacuum desiccator. Get 10 mg of crude product, analyze the content in it of the target nonapeptide, which is 95% (according to HPLC). Amino acid composition: Asp 0.97 (1), Glx 2.10 (2), Thr 2.80 (3), Lys 2.00 (2), Pro were not determined.

A solution of 0.54 g (1 mmol) of tripeptide (II) (4-fold excess with respect to the amino groups on the polymer) in 4 ml of NMP and 0.76 g (1 mmol) of “complex F” is added to the nonapeptidylpolymer (V). After 8 hours, the peptidyl polymer was filtered off, washed with NMP and DCM, and dried. The completeness of the attachment of the carboxyl component is determined using the ninhydrin test [4].

The final release and cleavage of the dodecapapeptide from the polymer is carried out in stage 1 by treating the corresponding dodecapapeptidyl polymer with a mixture of 10 ml of TFA, 0.25 ml of H ₂ O and 0.25 ml of TIBS for 1 h. Then the polymer is filtered off, washed with 2 × 2 ml of the deblocking mixture, the filtrate is evaporated and dry ether is added to the residue. The precipitate is filtered off, washed with dichloromethane (3 × 3 ml), ether (3 × 5 ml), dried in a vacuum desiccator. Obtain 0.6 g of crude product I, containing according to HPLC 90% of the target peptide.

After that, the peptide is purified using preparative HPLC on a Beckman device (USA), a 130T Diasorb-C16 column (2.5 × 250 mm) is used, the sorbent particle size is 10 μm. The eluents used are: buffer A — 0.01 M solution of ammonium acetate and buffer B — 80% acetonitrile in water, elution is carried out with a gradient of 0.5% per minute of buffer B from 100% buffer A, and a flow rate of 10 ml / min. Peptides are detected at a wavelength of 220 nm. Fractions containing the desired product are combined and lyophilized. The result is 0.29 g (75% based on the starting amino acid attached to the polymer carrier) of dodecapeptide I acetate. The homogeneity of the product, determined by analytical HPLC, is 98%. Amino acid composition: Thr 2.88 (3), Asp 2.03 (2), Glx 2.02 (2), Leu 1.02 (1), His 1.00 (1), Lys 2.00 (2), Pro were not determined. ¹ H-NMR (DMSO-d ₆ , δ, ppm): The spectrum contains signals (ppm):

1. Aspartic acid - 8.18 (NH ₂ , 2H), 4.13 (α-CH, 1H), 2.68, 2.80 (β-CH ₂ , 2H)

2. Histidine - 8.73 (NH, 1H), 4.67 (α-CH, 1H), 3.0, 3.09 (β-CH ₂ , 2H), 8.97 (C ₂ H, 1H, imidazole), 7.35 (C ₄ H, 1H imidazole)

3. Leucine - 8.18 (NH, 1H), 4.32 (α-CH, 1H), 1.45 (β-CH ₂ , 2H), 1.58 (γ-CH, 1H), 0.87, 0.84 (δ ', δ "-CH ₃ , 6H)

4. Aspartic acid - 8.43 (NH, 1H), 4.58 (α-CH, 1H), 2.72, 2.51 (β-CH ₂ , 2H)

5. Lysine - 7.79 (NH, 1H), 4.25 (α-CH, 1H), 1.64.1.50 (γ-CH ₂ , 2H), 1.28 (γ-CH ₂ , 2H), 1.51 (δ-CH ₂ , 2H) ), 2.74 (ε-CH ₂ , 2H)

6. Glutamine - 8.14 (NH, 1H), 4.31 (α-CH, 1H), 1.90, 1.78 (β-CH ₂ , 2H), 2.13 (γ-CH ₂ , 2H)

7. Threonine - 7.81 (NH, 1H), 4.22 (α-CH, 1H), 4.01 (β-CH, 1H), 1.02 (γ-CH ₃ , 3H)

8. Glutamine - 7.87 (NH, 1H), 4.38 (α-CH, 1H), 1.87, 1.72 (β-CH ₂ , 2H), 2.11 (γ-CH ₂ , 2H)

9. Threonine - 8.05 (NH, 1H), 4.37 (α-CH, 1H), 3.84 (β-CH, 1H), 1.13 (γ-CH ₃ , 3H)

10. Proline - 4.37 (α-CH, 1H), 2.03 (β-CH ₂ , 2H), 1.93, 1.84 (Y-CH ₂ , 2H), 3.72, 3.64 (δ-CH ₂ , 2H)

11. Lysine - 8.06 (NH, 1H), 4.32 (α-CH, 1H), 1.70 (β-CH ₂ , 2H), 1.36 (γ-CH ₂ , 2H), 1.52 (δ-CH ₂ , 2H), 2.75 (ε-CH ₂ , 2H)

12. Threonine - 7.65 (NH, 1H), 4.18 (α-CH, 1H), 4.14 (β-CH, 1H), 1.04 (γ-CH ₃ , 3H)

Mass spectrum, m / z: 1411.9 [M + H] ⁺ , gross formula: C ₅₉ H ₉₈ N ₁₈ O ₂₂ , calculated 1411.5.

As can be seen from the above examples, the claimed method can significantly increase the yield of the target product and simplify the technology for its production by reducing the stages of the process.

Information sources

1. DAN, 2005, Volume 404, No. 4, pp. 251-255.

2. Patent of the Russian Federation No. 2260598, published. 2005 (prototype).

3. Gershkovich A.A., Kiberev V.K. Chemical synthesis of peptides. Kiev: Naukova Dumka, 1992, pp. 4-10.

4. J. M. Stewart, J. D. Yang Solid Phase Peptide Synthesis Sec. Ed. Rockford USA: Pierce Chem.Com. 1984, p. 105.

Claims (3)

1. A method of producing a dodecapapeptide of the formula I H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH by the solid phase method by sequentially expanding the peptide chain starting from the C-terminal amino acid covalently linked to the polymer matrix, followed by processing the obtained dodecapapeptidyl polymer with a deprotecting agent for cleaving the protective groups and the polymer matrix and isolating the final product by HPLC, characterized in that the C-terminal nonapeptidyl polymer V is condensed with the protected tripeptide of formula II X-Asp (Y) -His-Leu-OH, where X, Y are protective groups. 2. The method according to claim 1, characterized in that X is a tert-butyloxycarbonyl group (Boc), Y is a tert-butyl group (Bu ^t ). 3. The tripeptide of formula II Boc-Asp (OBu ^t ) -His-Leu-OH, where Boc is tert-butyloxycarbonyl, Bu ^t -tert-butyl as an intermediate to obtain the dodecapapeptide of formula I H-Asp-His-Leu-Asp -Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH.

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