Method of obtaining dodecapeptide and tripeptide for its realisation

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.

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

H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH (I), and Tripeptide Boc-As(OBut)-His-Leu-OH formula (II), which is an intermediate compound in the synthesis.

Dodecapeptide formula I has the ability to inhibit the migration promonocytic cells TNR-1 and isolated from human blood monocytes under conditions of cell cultivation, as well as the migration of monocytes and granulocytes in inflammation in animals and can be used as a means to prevent the development of acute coronary conditions and prevention of restenosis after angioplasty and stenting, as well as acute inflammation and exacerbation of various chronic diseases such as gout and rheumatoid arthritis [1].

A known method of producing dodecapeptide formula I of the solid-phase method by stepwise extension of the peptide chain, starting with the C-terminal amino acids, covalently associated with a polymeric matrix, using the appropriate Fmoc-amino acid in the presence of a condensing agent, followed by the release of the final dodecapeptide in 2 stages: treatment with a solution of piperidine and then triperoxonane acid and subsequent purification of the obtained PR is product by high performance liquid chromatography (HPLC) [2]. It dodecapeptide formula I with a yield of 46% (based 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 a multi-stage process. The disadvantage is the use of expensive and difficult-derived amino acids in the stages of building up the peptide chain, starting with the C-terminal nonperformer (V).

These shortcomings make the method unsuitable for large-scale synthesis of dodecapeptide formula I.

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

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

carried out by successive growth of the peptide chain, starting with the C-terminal amino acids, covalently associated with a polymeric matrix, to obtain C-terminal nonparticipation (V), which is then condensed with a protected N-terminal Tripeptide of formula II:

where X, Y, - protective group, then the received dodecapeptide process debateroom the agent in stage 1 otscheplaut all protective groups and the polymer matrix and produce a final product with what omashu HPLC (see Annex 2, the inventive method).

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

List of abbreviations:

Asón - acetic acid;

Boc - tert-butyloxycarbonyl;

Buttert-butyl;

DIC - N,N1-diisopropylcarbodiimide;

DCM is dichloromethane;

DMF - N,N-dimethylformamide;

DMSO - d6 - deuterated dimethylsulfoxide;

Fmoc - 9-fluorenylmethoxycarbonyl;

HONSu - N-hydroxysuccinimide;

NAVT - 1-hydroxybenzotriazole;

HONp - para-NITROPHENOL;

NMP - N-organic;

NMM is N-methylmorpholine;

Pip - piperidine;

TIBS - triisobutylene;

TFA - triperoxonane acid;

HPLC - high performance liquid chromatography;

TLC is thin layer chromatography;

"complex of F is the set of pentafluorophenol with dicyclohexylcarbodiimide in the ratio of 3:1.

In the present method used derivatives of L-amino acids company Bachem (Switzerland), DIC, HONSu, HONp, NMP, TFA company Fluka (Switzerland). DMF was purified by distillation over ninhydrin and barium oxide. Analytical HPLC was carried out on the chromatograph company Gilson, France)was used column Ultrasphere ODS, 5 μm (4,6×250 mm) Beckman (USA)as the eluents used the buffer And 0.05 M KN2PO4at pH 3.0, buffer B was 70% acetonitrile in buffer A, elution with a concentration gradient from 0% to 60% buffer B in buffer And 40 min flow Rate 1 ml/min, detection at 220 nm. The individuality of the obtained compounds was confirmed by TLC on chromatographic plates Kiselgel 60 (Merck, Germany) in the solvent system: 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). Substances on the chromatograms showed chloro - benzidine and ninhydrin. The melting points (uncorrected) were determined on the device Boetius (Germany). Amino acid analysis of peptides, hydrolyzed 6 N. hydrochloric acid containing 2% phenol at 110°C for 24 h, were performed on the device Biotronik LC 5001 (Germany).1H-NMR spectra were taken on the spectrometer WH-500 Broker 500MHz (Germany) in DMSO-d at 300K, the concentration of the peptides was 2-3 mg/ml Chemical shifts were measured relative to tetramethylsilane. Mass spectra were recorded on the instrument PC-Kompact MALDI (Kratos, UK).

Synthesis of intermediates

Example 1. Boc-Asp(OBut)-His-Leu-OH (II):

9.52 g (20 mmol) of Boc-His(Boc)-ONp are dissolved in 100 ml of DMF, cooled to -10° With and added 2.6 g (20 mmol) of leucine in 10 ml of 2n. NaOH and stirred at 20°within 4 hours of the Completion of the reaction is controlled by TLC in system 1. The reaction mixture is evaporated, the oily residue is dissolved in 200 ml of water and extracted with ether (3×50 ml). The aqueous phase is acidified with citric acid to pH 4, extracted by ethylacetate (3×100 ml), combined an ethyl acetate extract is washed with water (3×50 ml), the solvent is removed in vacuum, the oily product is triturated with hexane. End up with 7.5 g (80%) of compound III. Rf0.48 (1), 0.83 (2). The resulting material is crystallized from a mixture of ether-hexane in the form of a corresponding dicyclohexylammonium salt. So pl. 118-122°C.

For removal of the BOC-protection of 6.0 g (12.8 mmol) of the compound (III) is dissolved in 30 ml of TFA and allowed to stand for 1 h at 20°C. Triperoxonane acid evaporated and triptorelin H-His-Leu-OH precipitated with ether. In the end you get a 6.0 g (95%) of compound IV. Rf0.27(3), 0.32 (4), 0.13 (5).

6.0 g (12.2 mmol) of H-His-Leu-OH·TFA (IV) is dissolved in 100 ml of DMF, the resulting solution was added 2.7 ml (24.4 mmol) of NMM and 4.71 g (12.2 mmol) of Boc-Asp(OBut)-ONSu. The reaction mixture was kept for 18 hours, after the reaction is controlled by TLC in system 2. After the reaction solution was evaporated, the residue is dissolved in 250 ml of a mixture of ethyl acetate and N.-butanol (4:1), add 1 equivalent (12.2 mmol) of hydrochloric acid, washed with water (3×70 ml) and evaporated. To the residue was added 100 ml of ether, the precipitated precipitate is filtered off, washed with ether (3×50 ml) and dried. End up with 5.4 g (82%) of compound II. Rf0.45 (2), 0.75 (3), 0.82 (4). TPL 145-147°C.1H-NMR spectrum contains signals (ppm):

1. Aspartic acid - 7.11 (NH2, 2H), 4.26 (αCH, 1H), 2.60, 2.38 (β-CH22N)

2. Histidine - 8.04 (NH, 1H), 4.59(α-CH, 1H), 3.12, 2.96 (βCH2, 2H), 8.96 (C2H, 1H, imidazole), 7.31 (C4H, 1H, imidazole)

3. Leucine - 8.16 (NH, 1H), 4.21 (αCH, 1H), 1.53 (βCH2, 2H), 1.62 (γCH, 1H), 0.88, 0.83 (δ',δ"CH3, 6N), and 1.37 (Vos, But, 18H)

Example 2.

2a. H-Asp(OBut)-Lys(Boc)-Gln(Trt)-Thr(But)-Gln(Trt)-Thr(But)-Pro-Lys(Boc)-Thr(But)-polymer (V)

For solid-phase synthesis using a copolymer of styrene with 1% divinylbenzene with hydroxymethoxypethidine anchor group containing 0.67 mmol/g) Fmoc-Thr (But) with a particle size 200-400 mesh company Bachem (Switzerland). Synthesis of nonparticipation carried out on the basis of 0.37 g (0.25 mmol) of Fmoc-Thr(But)-polymer in automatic mode on the peptide synthesizer, Applied Biosystems 431 And the standard program for a single condensation of Fmoc-amino acids.

To block functional groups of the side chains of the amino acids p is menaut following protections: tert-boutelou to carboxyl groups of aspartic acid and hydroxyl functions threonine; tert-butyloxycarbonyl (Vos) - protection for ε-amino group of lysine; trailing (Trt) group carboxamides functions of glutamine.

Protocol for solid-phase synthesis
No.OperationReagentProcessing time
1Flushing5×NMP3 min
2Release α-amino group20% Pip/NMP10 min
3Flushing5×NMP3 min
Activation1 mmol of Fmoc-amino acids +1 mmol HOBt+1 mmol of DIC in NMP20 min
6Condensation1 mmol of activated derivative Fmoc-amino acid in NMP90 min
7Flushing5×NMP3 min

To assess the quality of the intermediate V - C-terminal nonparticipation - conduct release and cleavage of the corresponding nonapeptide from the polymer carrier, using a sample (50 mg) intermediate V Sample V is treated with 5 ml TFA containing 0.1 ml of deionized water and 0.1 ml of triisobutylene for 1 h, the polymer is filtered off, the filtrate evaporated, the residue was added ether and the precipitate is filtered off, washed with dichloromethane (3×1 ml), ether (3 x 1 ml), then dried in a vacuum desiccator. Receive 10 mg of crude product, analyze the content 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 is not defined.

To nonparticipation (V) was added a solution of 0.54 g (1 mmol) Tripeptide (II) (4-fold excess relative to the amine groups on the polymer) in 4 ml of NMP and 0.76 g (1 mmol) of the complex F". After 8 hours participalion filtered off, washed with NMP and DCM and dried. Completeness attach carboxyl component is determined using a test with ninhydrin [4].

The final release and removal dodecapeptide from the polymer is carried out in stage 1 by treatment of the corresponding dodecapeptide a mixture of 10 ml of TFA, 0.25 ml of N2Oh and 0.25 ml TIBS within 1 hour the polymer was Then filtered off, washed with 2×2 ml deblokiruyuschee mixture, the filtrate is evaporated and to the residue was added dry ether. 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.

Then spend the purification of the peptide by using preparative HPLC on a Beckman instrument (USA), use column diasorb-C16 T (2.5×250 mm), once the er particles of sorbent - 10 μm. As eluents used: buffer A - 0,01M solution of ammonium acetate and buffer B was 80% acetonitrile in water, the elution is conducted with a gradient of 0.5% per minute buffer B to 100% buffer a, flow rate 10 ml/min, the Peptides detected at a wavelength of 220 nm. The fractions containing the desired product are combined and lyophilized. End up with 0.29 g (75% calculated on the starting amino acid attached to the polymeric carrier) acetate dodecapeptide I. the homogeneity of the product was determined using analytical HPLC, 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 is not defined.1H-NMR (DMSO-d6that δ, ppm): spectrum contains signals (ppm):

1. Aspartic acid - 8.18 (NH2, 2H), 4.13 (αCH, 1H), 2.68, 2.80 (βCH22N)

2. Histidine - 8.73 (NH, 1H), 4.67 (α-CH, 1H), 3.0, 3.09 (β-CH2, 2H), 8.97 (C2H, 1H, imidazole), 7.35 (C4H, 1H, imidazole)

3. Leucine - 8.18 (NH, 1H), 4.32 (α-CH, 1H), 1.45 (β-CH2, 2H), 1.58 (γ-CH, 1H), 0.87, 0.84 (δ',δ"CH3, 6N)

4. Aspartic acid - 8.43 (NH, 1H), 4.58 (α-CH, 1H), 2.72, 2.51 (β-CH22N)

5. Lysine - 7.79 (NH, 1H), 4.25 (α-CH, 1H), 1.64,1.50 (γCH2, 2H), 1.28 (γCH2, 2H), 1.51 (δCH2, 2H), 2.74 (εCH22N)

6. Glutamine - 8.14 (NH, 1H), 4.31 (α-CH, 1H), 1.90, 1.78 (β-CH2, 2H), 2.13 (γ-CH2, 2H)

7. Threonine - 7.81 (NH, 1H), 4.22 (α-CH, 1H), 4.01 (&x003B2; -CH, 1H), 1.02 (γCH3, 3H)

8. Glutamine - 7.87 (NH, 1H), 4.38 (α-CH, 1H), 1.87, 1.72 (βCH2, 2H), 2.11 (γCH22N)

9. Threonine - 8.05 (NH, 1H), 4.37 (α-CH, 1H), 3.84 (β-CH, 1H), 1.13 (γCH3, 3H)

10. Proline - 4.37 (α-CH, 1H), 2.03 (βCH2, 2H), 1.93, 1.84 (Y-CH2, 2H), 3.72, 3.64 (δCH22N)

11. Lysine - 8.06 (NH, 1H), 4.32 (α-CH, 1H), 1.70 (βCH2, 2H), 1.36 (γCH2, 2H), 1.52 (δCH2, 2H), 2.75 (εCH22N)

12. Threonine - 7.65 (NH, 1H), 4.18 (α-CH, 1H), 4.14 (β-CH, 1H), 1.04 (γCH3, 3H)

Mass spectrum, m/z: 1411.9 [M+H]+gross formula: C59H98N18O22calculated 1411.5.

As seen from the above examples, the claimed method can significantly improve the yield of the target product and to simplify the technology of its receipt by reducing process steps.

Sources of information

1. DAN, 2005, vol 404, issue 4, s-255.

2. The patent of Russian Federation №2260598, published. 2005 (prototype).

3. Herskowitz A.A., Kibirev VK Chemical synthesis of peptides. Kiev: Naukova Dumka, 1992, p.4-10.

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

1. The method of producing dodecapeptide formula I

H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH solid-phase method by successive growth of the peptide chain, starting with the C-terminal amino acids, covalently associated with a polymeric matrix, the next is th processing the received dodecapeptide debateroom agent for removal of protective groups and the polymer matrix and selection of the final product by HPLC, characterized in that the leaf nonparticipation V condense with the protected Tripeptide of the formula II X-Asp(Y)-His-Leu-OH, where X, Y is a protective group.

2. The method according to claim 1, characterized in that X represents a tert-butyloxycarbonyl group (Boc), Y - tert-boutelou group (But).

3. The Tripeptide of formula II Boc-Asp(OBut)-His-Leu-OH, where Boc is tert-butyloxycarbonyl, But-tert-butyl as intermediate compounds for obtaining dodecapeptide formula I H-Asp-His-Leu-Asp-Lys-Gln-Thr-Gln-Thr-Pro-Lys-Thr-OH.



 

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