Peptides possessing cytoprotective activity

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry, more specifically to new biologically active peptides which can be used in pharmacology and medicine to create new drug preparations that have cytoprotective activity.

EFFECT: new biologically active peptides are presented.

12 ex, 28 dwg, 2 tbl

 

The invention relates to organic chemistry, specifically to new biologically active peptides of the General formula:

A-L-Asp-L-Glu-B

where A-H - amino acid residue or acyl;

B-OH - residue amino acid, or any organic compounds that can form an amide or ester chemical bond with cytoprotective activity, which can find application in pharmacology and medicine at the creation of new medicines.

Known for a series of short peptides containing in its structure a derivative of the dipeptide Y-L-Glu-L-Asp-X and having the property of intercellular mediators [1-5]. The disadvantage of these compounds is their chemical instability and tendency to spontaneous rearrangements.

If X is a residue of glycine, there is a spontaneous migration of the remainder of the α-carboxylic group of aspartic acid to β-carboxyl. Although this transformation may occur in the case of any amino acid, the rate of this reaction strongly depends on the steric interaction [6-7]. If X is the residue of Proline or any other organic compound with a secondary amino group, it is spontaneous rupture of the connection between these residues [8].

It should be noted that these transformations lead to partial racemization of aspartic acid residue.

The purpose of the claimed technical the CSOs solution is to obtain a chemically stable peptides having cytoprotective activity. This goal is achieved by the described peptides of General formula:

A-L-Asp-L-Glu-B

where A-=H - residue amino acid or acyl;

B-=HE - residue amino acid, or any organic compounds that can form an amide or ester chemical bond.

The synthesis of the claimed compounds is carried out using generally accepted in the chemistry of biologically active peptides methods [9-10] and can be easily scaled up for commercial production.

The claimed compounds may find application in food, cosmetic or pharmaceutical industry. For substances used in these areas, one of the main criteria of applicability is the chemical stability during storage, and stability in the composition of the final products. Accepted abbreviations:

Boc - tert-butyloxycarbonyl

Bzl is benzyl

Z - benzyloxycarbonyl

Bu - tert-butyl

DCHA - DICYCLOHEXYL amine

TFA - triperoxonane acid

ONP - para-nitrophenyl

ONSu - N-oxyskandal

NMM is N-methylmorpholine

DMF - dimethylformamide

Tos - para-toluensulfonate

DCHA - dicyclohexylamine

Ala (a) - residue alanine

Asp (D) residue aspartic acid

Glu (E) residue glutamic acid

Lys (K) residue lysine

Gly (G) residue of glycine

rg (R) - the residue arginine

Trp (W) residue tryptophan

Leu (L) - leucine residue

DMEM - Dulbecco''s Modified Eagle's Medium, medium Needle, modified, Dulbecco

FBS - fetal bovine serum, serum fetal cow

PBS - phosphate buffer saline, phosphate saline buffer, pH 7,4

HeLa (ATCC Number CCL-2,2, subclone S3) - cell adenocarcinoma of the uterus human epithelial morphology

NIH 3T3 (ATCC Number CRL-1658) - embryonic fibroblasts mouse

DMSO - dimethyl sulfoxide

EDTA-ethylenediaminetetraacetate

The invention is illustrated by the following examples:

Example 1. H-Asp-Glu-OH (DE)

1. Boc-Asp(OBzl)-Glu (OBzl)-OBzl_

To a cooled to -20°C solution of 48.5 g (150 mmol) of Boc-Asp (Bzl)-OH in 300 ml of DMF was added to 16.5 ml (150 mmol) of NMM and 19.5 ml (150 mmol) of isobutyronitrile and was stirred for 15 minutes and the Mixture was stirred at the same temperature for 20 minutes Then was added, cooled to -20°C a solution of 74.5 g (155 mmol) aminocompounds H-Glu(Bzl)-OBzlTos in 200 ml of DMF containing 17 ml of NMM (155 mmol). The reaction mixture was stirred for 1 hour at -10°C, for 2 hours at room temperature. Was evaporated, the residue was dissolved in ethyl acetate (500 ml) and sequentially washed with 5% NaHCO3H2O, 2% H2SO4N2On (twice 200 ml each), evaporated, re-evaporated with isopropyl alcohol. To the resulting oil was added 500 ml of hexane and 50 ml of ether, and the resulting oil was rubbed out before formation of the solid is ASTIC. The product crystallized in the refrigerator. The precipitate was filtered, washed on the filter with hexane, and dried. Received 89 g (89%) of chromatographically homogeneous product.

2. H-Asp-Glu-OH

12,64 g (20 mmol) of the protected dipeptide Boc-Asp(Bzl)-Glu(Bzl)-OBzl was dissolved in 100 ml of chloroform and the resulting solution at 0°C was added 100 ml triperoxonane acid, the mixture was stirred for 1 hour at room temperature and the solvent was evaporated in vacuum. The residue was dissolved in 300 ml ethyl acetate and washed with 5% solution of bicarbonate 3 times 200 ml of ethyl Acetate was evaporated, the residue was dissolved in 500 ml of 80% acetic acid and was first made over 5% Pd/C at room temperature. After completion of the hydrogenation (control by TLC Rf system: chloroform: methanol: 32% acetic acid 60:45:20) the catalyst was filtered, the filtrate was evaporated, the residue was dissolved in 300 ml of water, was re-evaporated to half volume and liofilizovane. Received 4.8 g (91%) of the target dipeptide. According to HPLC the purity of the product is more than 95%.

Example 2. H-Ala-Asp-Glu-OH (ADE)

1. Z-Ala-Asp(OBzl)-Glu(OBzl)2

19 g (30 mmol) of Boc-Asp(OBzl)-Glu(OBzl)2was dissolved in 200 ml of TFA, 1 hour, evaporated, the residue was dissolved in 500 ml of dry DMF and added portions of Na2CO3before the termination of the allocation of CO2. The precipitate was filtered off, washed with 100 ml of DMF, was added to 3.33 ml (30 mmol) N-methylmorpholine and 9,72 g (30 mmol) of Z-Ala-ONSu and left the and 15 hours at room temperature. The reaction mixture was evaporated at 80%, the resulting syrupy residue was added 800 ml of isopropyl alcohol was stirred for 2 h at +4°C, the fine precipitate was filtered, washed with isopropyl alcohol, ether, and dried. The output of 20.3 g (92%) of product.

2. H-Ala-Asp-Glu-OH

of 20.3 g (28 mmol) of the protected Tripeptide Z-Ala-Asp(OBzl)-Glu(OBzl)2when heated to +50°C was dissolved in 1000 ml of Asón + 100 ml of water and was first made over 5% Pd/C, periodically stirring the reaction mixture. The course of the reaction was controlled by TLC. After the catalyst was filtered, the filtrate was evaporated to the consistency of liquid oils and added 800 ml of isopropyl alcohol. The precipitate was filtered, washed with isopropyl alcohol, ether, and dried. The substance was dissolved in deionized water, was evaporated residues of isopropyl alcohol and liofilizirovanny. Output to 7.67 g (85%) of product. HPLC over 95%.

In the spectrum of TMR:

Ala - 3,81 (α-CH); 1,35 (β-CH3); 8,08 (NH2)

Asp 4,62 (α-CH); 2,70; 2,53 (β-CH2); 8,63 (NH)

Glu - 4,20 (α-CH); 1,97; 1,78 (β-CH2); and 2.26 (γ-CH2); 8,19 (NH).

Example 3. H-Lys-Asp-Glu-OH (KDE)

1. Z-Lys (Z)-Asp(OBzl)-Glu(OBzl)2

19 g (30 mmol) of Boc-Asp(OBzl)-Glu(OBzl)2(1) was dissolved in 200 ml of TFA, over an hour was evaporated, the residue was dissolved in 500 ml of dry DMF and added portions of Na2CO3before the termination of the allocation of CO2. The precipitate was filtered, probyvala ml of DMF, added to 3.33 ml (30 mmol) N-methylmorpholine and 15,36 g (30 mmol) of Z-Lys(Z)-ONSu and left at room temperature for 15 hours. The reaction mixture was evaporated, the resulting syrupy residue was dissolved in 300 ml ethyl acetate and washed successively with 2% sulfuric acid, with water until neutral, 5% soda solution and again with water. The ethyl acetate was evaporated and the oily residue was added 500 ml of isopropyl alcohol was stirred for 2 h at +4°C, the precipitate was filtered, washed with isopropyl alcohol, ether, and dried. The output of 21.6 g (90%) of product.

2. H-Lys-Asp-Glu-OH

of 21.6 g (27 mmol) of the compound Z-Lys (Z)-Asp(OBzl)-Glu(OBzl)2obtained as described above was dissolved by heating in 0.5 l of glacial acetic acid was added 50 ml of water and was first made in the presence of 5 g of palladium catalyst. The catalyst was filtered and evaporated the solvent. Was led from a mixture of acetic acid-ethanol. If necessary, the product was then purified by ion exchange chromatography on a column of SP-Sephadex, put in water, wash 0.04 M pyridine-acetate buffer (pH of 5.4). The fractions containing the desired compound were evaporated. Syrupy residue was dissolved in 300 ml of water, was evaporated to half volume, and treated with activated carbon and was liofilizovane. The output of 8.95 g (85%) of product. Product purity according to HPLC, more than 95%.

In the spectrum of TMR:

Lys - 3,76 (α-CH);1,72 (β-CH 2); 1,36; 1,52 (γ-CH2); 2,74 (ε-CH2); 8,11 (NH2)

Asp 4,63 (α-CH); 2,71; 2,54 (β-CH2); 8,68 (NH)

Glu - 4,19 (α-CH); 1,98; 1,78 (β-CH2); 2,28 (γ-CH2); 8,31 (NH).

Example 4. H-Asp-Glu-Pro-OH (DEP)

1. Z-Asp(OBut)-Glu(OBut)- OH·DCHA

17.5 g (100 mmol) of H-Glu(OBut)-OH was dissolved (suspended) in methanol was added 50 ml of 40% Triton b In methanol and after complete dissolution was evaporated in vacuum. The residue was dissolved in 300 ml dimethylformamide, was evaporated on a quarter and added 42,0 g (100 mmol) of Z-Asp(OBut)-ONSu. After 12 hours (control - TLC in the system chloroform: methanol: acetic acid 9:1:0.5 "B"), the reaction mixture was evaporated, the residue was dissolved in ethyl acetate, washed sequentially with 2% sulfuric acid, water, dried over Na2SO4that was evaporated. The residue was dissolved in 150 ml of ether, was added 20 ml (100 mmol) of dicyclohexylamine and left at room temperature until complete precipitation. The precipitate was filtered, washed with ether, dried in the air. The output 60 g (87%) of chromatographically uniform substance.

2. H-Asp-Glu-Pro-OH

28 g (40 mmol) of Z-Asp(OBut)-Glu(OBut)-HE·DCHA suspended in 500 ml of ethyl acetate, washed with 2% sulfuric acid, water, dried over Na2SO4the desiccant was filtered, the filtrate was evaporated, the residue was dissolved in 150 ml of dimethylformamide, cooled to -25°C, cooled solution was added to 4.4 ml (40 IMO the b & N methylmorpholine and 5.2 ml (40 mmol) of isobutylacetate, kept at -20°C for 15 min and added to cooled to -25°C a solution of 5.6 g (44 mmol) of N-hydroxysuccinimide 25°C in 50 ml of dimethylformamide. After 30 min the solution was added 10.6 g (44 mmol) H-Pro-OBzl HCl and 5 ml of N-methylmorpholine in 50 ml of dimethylformamide and left at room temperature overnight. The reaction mixture was evaporated, the residue was dissolved in ethyl acetate and washed in the usual way. The ethyl acetate was evaporated, the residue was evaporated with toluene. The resulting oil was dissolved in TFA, was kept for one hour, evaporated, the residue was treated with a mixture of ether - hexane 1:1, the precipitation was filtered, washed on the filter with a mixture of ether - hexane 1:1, hexane, dried in a desiccator over potassium.

The obtained product was dissolved in a mixture of ethanol - water - acetic acid (600:100:100) and was first made over 5% PdC. the Catalyst was filtered, the filtrate was evaporated. The residue was perioadele of acetic acid, isopropyl alcohol, filtered, washed on the filter with isopropyl alcohol, ether, and dried. Yield 12.2 g (85%). Purity according to HPLC, more than 95%.

In the spectrum of TMR:

Asp - 4,13 (α-CH); 2,79; 2,66 (β-CH2); 8,14 (NH2)

Glu - 4,59 (α-CH); 1.93 and 1,72 (β-CH2); 2,36 (γ-CH2); 8,67 (NH)

Pro - 4,23 (α-CH); 2,14; 1,84 (β-CH2); 1,90 (γ-CH2); 3,63 (δ-CH2).

Example 5. H-Asp-Glu - Arg-OH (DER)

1. Z-Glu(OtBu)-ArgOH

To a solution of 104,4 g (228 mmol) of Z-Glu(OtBu)ONP in 0.5 l of dimethylformamide PR is balali to 38.3 g (228 mmol) of H-Arg-OH. The reaction mixture was stirred at room temperature for 24 hours. Then kept at a temperature of -4°C for four hours, the precipitate was filtered and washed on the filter with 0.4 l of a mixture of dimethylformamide-ether (1:2), 0,45 liter of ether, 0.4 l of hexane. Was dried. Output: 91,7 g (82,3%) of chromatographically homogeneous Z-Glu(OtBu)ArgOH (control system: chloroform-methanol-acetic acid(5:3:1)).

2. H-Asp-Glu - Arg-OH

The solution of 90.6 g (185 mmol) ZGlu(OtBut)ArgOH in 1.2 l of ethyl alcohol was first made over 10 g of 5% Pd/C. After completion of the hydrogenation (control by TLC in the system: chloroform-methanol-acetic acid (5:3:1)catalyst was filtered, the solvent was evaporated. Received syrupy product was dissolved in 500 ml of dimethylformamide and the resulting solution was added 75,9 g (185 mmol) of BOC-Asp(OtBu)-ONP. The reaction mixture was left at room temperature for 15 hours to complete the reaction (control system: chloroform-methanol-acetic acid (5:3:1)). The solvent was evaporated, the resulting oil was dissolved in 150 ml of chloroform and applied on a column of silica gel (400 g), washed with chloroform before gathering NITROPHENOL, 10% ethanol in chloroform, washed 30% ethanol in chloroform, evaporated (control system: chloroform-methanol-acetic acid (5:3:1)). To the residue was added 400 ml triperoxonane acid and the mixture was stirred for 2 hours at room temperature is. The acid was evaporated, to the residue was added 1000 ml of diethyl ether, the resulting amorphous precipitate was filtered, washed on the filter with ether twice 150 ml) and dried. The substance was dissolved in 1500 ml of water, was evaporated to oil was re-dissolved in 1000 ml of water and applied on a column of SP-Sephadex. The column was washed with two volumes of 0.05 M pyridine-acetate buffer and washed substance the same 0.2 M buffer. The fractions containing the product were combined, the solvent was evaporated, re-evaporated with water to remove residual pyridine-acetate and liofilizovane. Received 65 g (75%) of product with a purity according to HPLC, more than 95%.

In the spectrum of TMR:

Asp - 4,16 (α-CH); 2,82; 2,65 (β-CH2); 8,14 (NH2)

Glu - 4,35 (α-SN) 1,93; 1,87 (β-CH2); 2,30 (γ-CH2); at 8.60 (NH)

Arg - 4,16 (α-CH); 1,75; 1,62 (β-CH2); 1,54; 1,50 (γ-CH2); 3,10 (δ-CH2); 7,58 (ε-NH2); 8,28 (NH).

Example 6. H-Asp-Glu-Gly-OH (DEG)

1. Boc-Glu (BzI)-Gly-Bzl

To a cooled to -20°C solution 8,43 g (25 mmol) of Boc-Glu (Bzl)-OH in 100 ml of DMF was added 2,77 ml (25 mmol) of NMM and 3.25 ml (25 mmol) of isobutylacetate, was stirred for 15 min and was added cooled to -20°C solution of 7.97 g (25 mmol) aminocompounds H-Gly-OBzl×Tos in 50 ml of DMF containing 3 ml of NMM (27 mmol). The reaction mixture was stirred for 1 hour at -10°C, for 2 hours at room temperature. Was evaporated, the residue was dissolved in ethyl acetate (200 ml) and posledovatel is but washed with 5% NaHCO 3N2Oh, 2% H2SO4H2O (twice 200 ml each), evaporated, re-evaporated with isopropyl alcohol. The product crystallizes from isopropyl alcohol-hexane 1:1 at 4°C in the refrigerator. The precipitate was filtered, washed on the filter with hexane, and dried. Obtained 11 g (91%) of chromatographically homogeneous product.

2. Z-Asp(Bzl)-Glu(Bzl)-Gly-Bzl

10 g (20,7 mmol) of Boc-Glu(OBzl)-Gly-OBzl was dissolved in 50 ml of TFA, over an hour was evaporated, the residue was dissolved in 100 ml of dry DMF and added portions of Na2CO3before the termination of the allocation of CO2. The precipitate was filtered off, washed with 50 ml of DMF, and to the resulting solution were added at 9.53 g (21 mmol) of Z-Asp(Bzl)-ONSu and left at room temperature for 15 hours. The reaction mixture was evaporated, the resulting syrupy residue was dissolved in 300 ml ethyl acetate and washed successively with 2% sulfuric acid, with water until neutral, 5% soda solution and again with water. The ethyl acetate was evaporated and the oily residue was added 100 ml of isopropyl alcohol was stirred for 2 h at +4°C, the precipitate was filtered, washed with isopropyl alcohol, ether, and dried. Yield 12 g (80%) of chromatographically homogeneous product.

3. H-Asp-Glu-Gly-OH

of 7.23 g (27 mmol) of Z-Asp(Bzl)-Glu(Bzl)-Gly-Bzl, obtained as described above was dissolved by heating in 0.5 l of glacial acetic acid was added 50 ml of water and was first made in outstay 5 g of palladium catalyst. The catalyst was filtered and the solvent was evaporated. The product was perioadele of a mixture of acetic acid-isopropyl alcohol. Received 3.0 g (94%). Product purity according to HPLC, more than 95%.

In the spectrum of TMR:

Asp - 4,14 (α-CH); 2,87; 2,68 (β-CH2); 8,16 (NH2)

Glu - 4,35 (α-SN) 1,94; 1,80 (β-CH2); 2,31 (γ-CH2); 8,63 (NH)

Gly - 3,75 (α-CH2); 8,28 (NH).

Example 7. H-Ala-Asp-Glu-Leu-OH (ADEL)

1. Boc-Glu(Bzl)-Leu-OBzl

To a solution of 26.6 g (78 mmol) of Boc-Glu(Bzl)-OH in 200 ml of dimethylformamide and 8,65 ml of N-methylmorpholine cooled to -15°C, was added 10.4 ml (80 mmol) of isobutylacetate, the mixture was stirred at this temperature for 20 min and was added cooled to -15°C a solution of 30.5 g (78 mmol) of H-Leu-OBzl*Tos, the pH of which had previously been brought to 8.5, and 9.0. After one hour the solvent was evaporated, the residue was dissolved in 500 ml ethyl acetate and washed successively with 400 ml of 2% sulfuric acid solution, water, 5% solution of soda and water. The organic layer was separated and the ethyl acetate was evaporated. The residue was led from a mixture of ether-hexane (1:1). Received of 37.8 g (90%) of chromatographically uniform substance.

2. Boc-Asp(Bzl)-Glu(Bzl)-Leu-OBzl

27 g (50 mmol) of Boc-Glu(Bzl)-Leu-OBzl was dissolved in 100 ml of chloroform and the resulting solution was added 100 ml triperoxonane acid. The mixture was stirred at room temperature for 2 hours and the solvent was evaporated. The residue was dissolved in 200 ml of dimethylformamide and portions of primav the Yali soda until after the allocation of CO 2. The precipitate was filtered off, washed with 50 ml of dimethylformamide, and the filtrate brought the pH to 8.5 and cooled to -20°C (Solution 1).

Simultaneously to a solution of 16.5 g (50 mmol) of Boc-Asp(Bzl)-OH in 100 ml of dimethylformamide was added 5.6 ml of N-methylmorpholine, the mixture was cooled to - 20°C and was added to 6.5 ml (50 mmol) of isobutylacetate. The mixture was stirred at this temperature for 30 min (Solution 2).

Maintaining the cooling, both solutions were mixed and left to mix to reach room temperature (approximately 2 hours). The solvent was evaporated, the residue was dissolved in 300 ml of ethyl acetate and then washed with 2% sulfuric acid solution, water, 5% solution of soda, water, and the solvent was evaporated. After crystallization from ether got 32,4 g (87%) of chromatographically homogeneous BOC-Asp(Bzl)-Glu(Bzl)-Leu-OBzl.

3. Z-Ala-Asp(Bzl)-Glu(Bzl)-Leu-OBzl

of 22.2 g (30 mmol) of Boc-Asp(Bzl)-Glu(Bzl)-Leu-OBzl was dissolved in 100 ml of chloroform and the resulting solution was added 100 ml triperoxonane acid. The mixture was stirred at room temperature for 2 hours and the solvent was evaporated. The residue was dissolved in 200 ml of dimethylformamide and portions were added soda until after the allocation of CO2. The precipitate was filtered off, washed with 50 ml of dimethylformamide, and the filtrate brought the pH to 8.5 and was added 9.6 g (30 mmol) of Z-Ala-ONSu. The reaction mixture was left for 15 hours at room temperature. The solvents pariva and, the residue was dissolved in 500 ml of ethyl acetate and then washed with 2% sulfuric acid solution, water, 5% solution of soda, water, and the solvent was evaporated. After crystallization from isopropyl alcohol received of 22.4 g (87,8%) of chromatographically homogeneous Z-Ala-Asp(Bzl)-Glu(Bzl)-Leu-OBzl.

4. H-Ala-Asp-Glu-Leu-OH

17 g (20 mmol) of the protected tetrapeptide Z-Ala-Asp(Bzl)-Glu(Bzl)-Leu-OBzl was dissolved in 300 ml of 80% acetic acid and was first made over 5% PdC at room temperature. After the reaction the catalyst was filtered off, washed on the filter with acetic acid and the solvent was evaporated. The residue was dissolved in hot water, cooled to room temperature and left at 4°C for 15 hours. The precipitation was filtered, dried in vacuum over KOH. Received 8 g (89%) of product with a purity according to HPLC, more than 95%.

In the spectrum of TMR:

Ala - 3,83 (α-CH); 1,34 (β-CH3); 8,08 (NH2)

Asp br4.61 (α-CH); 2,72; 2,54 (β-CH2); 8,68 (NH)

Glu - 4,28 (α-CH); 1,91; 1,76 (β-CH2); 2,23 (γ-CH2); 7,93 (NH)

Leu - 4,17 (α-CH); 1,52 (β-CH2); 1,74; 1,62 (γ-CH2); 0,83 (δ-CH3); 0,89 (δ-CH3); 8,12 (NH).

Example 8. H-Asp-Glu-Leu-OH (DEL)

14.9 g (20 mmol) of the protected Tripeptide Boc-Asp(Bzl)-Glu(Bzl)-Leu(Bzl) was dissolved in 50 ml of chloroform and the resulting solution was added 50 ml triperoxonane acid. The mixture was stirred for 1 hour at room temperature and the solvent was evaporated. The residue was dissolved in 300 ml of 0% acetic acid and was first made over 5% PdC at room temperature. After the reaction the catalyst was filtered off, washed on the filter with acetic acid and the solvent was evaporated. To the residue was added 200 ml of isopropyl alcohol. The precipitation was filtered, dried in vacuum over KOH. Received 7 g (93%) of product with a purity according to HPLC, more than 95%.

In the spectrum of TMR:

Asp - 4,13 (α-CH); 2,81; 2,65 (β-CH2); 8,15 (NH2)

Glu - 4,34 (α-CH); 1,94; 1,80 (β-CH2); 2,30 (γ-CH2); 8,58 (NH)

Leu - 4,19 (α-CH); 1,52 (β-CH2); 1,53; 1,62 (γ-CH2); 0,83 (δ-CH3); 0,89 (δ-CH3); by 8.22 (NH).

Example 9. H-Lys-Asp-Glu-Trp-NH2(KDEW-NH2)

1. Z-Glu(OtBu)-Trp-NH2

To a solution of 35.4 g (100 mmol) of Z-Glu(OtBu)-OH in 200 ml of dimethylformamide and 11.1 ml of N-methylmorpholine cooled to -15°C, was added 13 ml (100 mmol) of isobutylacetate, the mixture was stirred at this temperature for 20 min and was added cooled to -15°C a solution of 20.4 g (100 mmol) of H-Trp-NH2, the pH of which had previously been brought to 8.5, and 9.0. After one hour the solvent was evaporated, the residue was dissolved in 500 ml ethyl acetate and washed successively with 400 ml of 2% sulfuric acid solution, water, 5% solution of soda and water. The organic layer was separated and the ethyl acetate was evaporated. The residue was led from a mixture of ether-hexane (1:1). Got to 45.2 g (86%) of chromatographically homogeneous Z-Glu(OtBu)-Trp-NH2.

2. Z-Asp(OtBu)-Glu(OtBu)-Trp-NH2

To a cooled to -20°C solution of 24.7 g (76.5 mmol) of Z-Asp (OtBu)-OH in 300 m is DMF) was added to 8.5 ml (76,5 mmol) NMM and 9,95 ml (765 mmol) iBuOCOCl and was stirred for 15 minutes The mixture was stirred at the same temperature for 20 minutes Then was added, cooled to -20°C the solution obtained by the hydrogenation of 40 g (76.5 mmol) of Z-Glu(OtBu)-Trp-NH2in 200 ml of DMF. The reaction mixture was stirred for 1 hour at -10°C, for 2 hours at room temperature. Completeness of the reaction was controlled in the system chloroform: methanol: acetic acid 9:1:0.5. The solvent was evaporated, the residue was dissolved in ethyl acetate (500 ml) and sequentially washed with 5% NaHCO3H2O, 2% H2SO4H2O (twice 200 ml each), evaporated, re-evaporated with isopropyl alcohol. The product was crystallized from a mixture of ethyl acetate-hexane (2:1) at 4°C in the refrigerator. The precipitate was filtered, washed on the filter with hexane, and dried. Received of 46.1 g (87%) of chromatographically homogeneous product.

3. Z-Lys(Z)-Asp(OtBu)-Glu(OtBu)-Trp-NH2

40 g (57.6 mmol) obtained as described above, the protected Tripeptide

Z-Asp(OtBu)-Glu(OtBu)-Trp-NH2was dissolved in 400 ml of ethanol and was first made over 5% PdC. After completion of the reaction the catalyst was filtered, the alcohol was evaporated, the residue was dissolved in 200 ml of dimethylformamide and the resulting solution was added 29.5 g (57.6 mmol) of Z-Lys(Z)-ONSu. The mixture was left for 15 hours at room temperature. The solvent was evaporated, the residue was dissolved in hot ethyl acetate and left to crystallize. Obrazovavshijsya was filtered, washed with a mixture of ethyl acetate-hexane (1:1), dried. Received 45 g (82%) of chromatographically uniform substance.

4. H-Lys-Asp-Glu-Trp-NH2

40 g (41,8 mmol) of Z-Lys(Z)-Asp (OtBu)-Glu(OtBu)-Trp-NH2was dissolved in 500 ml of ethanol, was added 5 g of 5% PdC in 50 ml of water and was first made before the end of the reaction. The catalyst was filtered, the solvent was evaporated. The residue was treated with 200 ml triptocaine acid containing 1% mercaptoethanol. After 1 hour, the acid was evaporated and to the residue was added 500 ml of ether. The precipitation was filtered, washed on the filter with ether, and dried. Was dissolved in 100 ml of water and applied on a column of SP-Sephadex (or similar cation-exchanger), balanced 0.05 M pyridine-acetate buffer. Washed with two volumes of starting buffer, one volume of 0.1 M buffer and the substance was washed with 0.2 M buffer. The fractions containing the desired product were combined, the solvent was evaporated, re-evaporated with 300 ml of water to remove traces of pyridine-acetate buffer. The residue was dissolved in 400 ml of water and was liofilizovane. Received 26 g (79%) khromatograficheskii homogeneous product with a purity according to HPLC, more than 95%.

In the spectrum of TMR:

Lys - 3,75 (α-CH), 1,70 (β-CH2)and 1.51; 1,34 (γ-CH2), 2,73 (ε-CH2), 8,12 (NH2)

Asp br4.61 (α-CH), 2,55; 2,72 (β-CH2), 8,73 (NH)

Glu - 4,19 (α-CH),1,86; 1,73 (β-CH2), 2,18 (γ-CH2), 8,16 (NH)

Trp - 4,43 (α-CH), 3,14, 2,98 (β-CH2), 784 (NH); 7,58; of 6.96; 7,05; 7,81 (CH aromatic); 10,8 (NH indole).

Example 10. Study of stability of peptides during storage.

The peptides were dissolved in 0.05 M acetylamino buffer with pH 4.5 and pH 7.5 in such a way that their concentration was approximately 1 mg/ml Solution was sterile filtered and stored in sterile, well-stoppered bottles. Upon expiration of the substance was analyzed by HPLC. The results are presented in table 1.

Table 1
Stability of peptides during storage
PeptideDuration (time, day)
1 hour10306090
Storage conditions (pH, 22°C)
4.57.54.57.54.57.54.57.54.5ADE97.297.297.1597.297.197.1397.197.0597.1596.9
DER98.198.198.198.197.997.798.0597.697.997.5
DEP96.596.596.596.4596.496.396.596.296.395.7
DEG96.796.796.796.796.696.596.396.296.2 95.8
ADEL98.298.298.398.298.198.0598.198.198.097.8

The results are presented in the form of the purity of the investigated products, expressed in percent.

Presents the results of the analyses indicate that the products are well kept even in solutions at different pH values.

Example 11. Study of biological activity of peptide drugs in standard cell culture

Study was made of the biological activity of the synthesized peptides in five different concentrations (400, 200, 100, 10 and 1 ng/ml), using as a model system of cultured cells of mouse and man: HeLa S3 and NIH T.

The biological activity of each peptide was determined by the following parameters: the effect of peptides on the proliferative activity of cells and the ability of peptides to induce apoptosis and necrosis. For this were investigated following indicators: the proportion of dead cells, the total number of cells in the vial, the level of apoptotic cell death.

The cultivation and processing the peptides

Cultivation of fibroblasts NIH ZTZ carried out in complete culture medium DMEM (Paneco, RF)containing 10% fetal serum (Paneco), gentamicin 2 u/ml (Paneco), glutamine 29,2 µg/ml (Paneco), in CO2-incubator. For cultivation of used vials firm SPL (South Korea) with an area of 25 cm2and 175 cm2. Re-seeding of the cells was carried out in a ratio of 1:5-1:7. Upon reaching approximately 50% of confluently vials was added to the peptides in the above final concentrations. In each experiment used two vials with the same concentration of each peptide. In the control vials was added an appropriate volume of complete medium without peptide. Each peptide is carried out by two independent experiment (two repetitions each). After 24 hours incubation with the peptides, the cells were removed from the culture vial with processing in a mixture of trypsin and versene (1:3, Paneco). Cells from each vial was placed in an equal volume of buffered saline (0.01 M PBS, pH of 7.2). Cell suspension was divided into 3 equal parts to study the above parameters.

The cultivation of HeLa cells were performed similarly except that the re-seeding of the cells was carried out in a ratio of 1:3-1:4.

A study of indicators was performed using flow cytometry. Used proton the th cytometer sorter FACS Vantage (Becton Dickinson, USA). Before each series of tests, the device was ustravel and calibrated according to standard microspheres (Polyscience) in accordance with the methodology recommended by the manufacturer.

Determination of the total number of cells in the vial and the fraction of dead cells

To the cell suspension obtained as described above was added propidium iodide (prodium iodide-PI, Sigma, USA) at a final concentration of 10 μg/ml and the calibration particles Calibrite (Becton Dickinson, USA)labeled with fluoresceinisothiocyanate (fitzum). The final concentration of the calibration particles was determined using the camera Goryaeva. Incubated for 10-12 min and immediately analyzed on a flow cytometer by 4 parameters - direct scattering, side scattering, fluorescence of PI fluorescence fitza. Each sample was analyzed in 20 testlets. The data recorded in the file. Then there was a computer processing data using the CellQuestPro software (Becton Dickinson, USA).

Determined the percentage of PI+cells with damaged plasma membranes (dead cells). The level of destruction in the control bottles (15-20 pieces) were averaged and taken as 100%. The possible influence of the peptides was assessed by the ratio control.

Each file was found FITZ+calibration particles and determine their number. Then in terms of light scattering identified cells and found their number in t is x the same files. Expected ratio of cells/calibration particles in each file (the sample), which were judged on the number of cells in the vial. The ratio of cells/calibration particles in the control was taken as 100%. The number of cells in the experimental bottles was estimated percentage compared to control.

Determination of cell death by apoptosis

The method is based on the fact that in cells undergoing apoptotic death, appears fragmented DNA, which is removed from the cells during treatment with HCl solution. Then remaining in the DNA is stained with specific dyes (e.g., PI) and define detailed ' cell ' of the DNA content. Apoptotic cell death is assessed according to the percentage of cells with hypodiploid DNA content.

Cell suspension obtained as described above was cooled to +4°C. the cell suspension was added to the cooled ethanol in a volume ratio of 1:3 with constant stirring. The cells were kept for 1-2 weeks before analysis. By centrifugation remove the ethanol from the sample, then deleted the fragmented DNA of cells with 0,6M HCl (37°C, 10 min). Next, the cells were washed by PBS and were stained with 0.3 ml of PI (50 μg/ml) in PBS, kept for 15 min at room temperature and analyzed on a flow cytometer. PI in this concentration allows the determination of the detailed ' cell ' of DNA content and views the ü fraction of cells with fragmented DNA with high resolution. To measure the fluorescence of PI was using narrowband filters 585/42 nm, the laser power was 57 mW. Each sample was analyzed 5·103cells, and then performed computer processing using CellQuestPro software (Becton Dickinson, USA). At the first stage of the analysis identified a region of cells according to the scattering, then in this region were evaluated by fluorescence of PI. Marker hypodiploid of DNA content exhibited on reference samples. The results are shown in Fig.1 - 12.

Example 12. The study of protective properties of peptides

In experiments to study the effects of peptides on apoptosis and necrosis of used reagents ApoTarget Annexin-V FITC Apoptosis Kit (Invitrogen, USA).

Apoptosis - programmed cell death, which is the result of the realization of the genetic program or a response to external factors and requiring expenditure of energy and the synthesis of macromolecules. Apoptosis is accompanied by the appearance of the characteristic cytological features (marker of apoptosis) and molecular processes. In living cells, in particular, the observed asymmetric distribution of the different phospholipids between the inner and outer monolayers of the cytoplasmic membrane: phospholipids containing choline, such as phosphatidylcholine and sphingomyelin, which are localized mainly in the outer monolayer, and the phosphatidylethanolamine and phosphatidylserine (aminoff the lipids) - in the interior. In the process of apoptosis cicoplafest membrane undergoes changes, one of which is the transfer of phosphatidylserine from the inner to her in the outer monolayer, where phosphatidylserine is available for binding with Annexin-V labeled with a fluorescent dye FITC. Annexin-V is a calcium-dependent, phospholipid-binding protein, mol. mass 35-36 kDa high affinity to phosphatidylcholine (Kdequal to 5×10-10M). Annexin-V-binding analysis based on rapid and highly specific detection of cells containing phosphatidylcholine in the outer monolayer of the cytoplasmic membrane, i.e. cells in early stages of apoptosis.

In addition to Annexin-V labeled with a fluorescent dye FITC, a set of reagents ApoTarget Annexin-V FITC Apoptosis Kit contains one dye - propidium iodide (PI). PI has the ability to penetrate only in those cells that have violated the integrity of the cytoplasmic membrane, which is one of the characteristic features of the late stages of apoptosis or necrosis. Penetrating into cells with damaged cell membrane, PI binds to DNA. Red fluorescence of the formed complexes are detected using a flow cytometer.

Thus, using the double staining cells (Annexin-V FITC plus PI) and the method of production cytofluorometry, once iChat 3 population of cells and to determine their percentage: 1) neuroprothesis cells (Annexin-V-negative and PI-negative), 2) cells at early stages of apoptosis (Annexin-V-positive and PI-negative), 3) necrotic cells or cells at the late stage of apoptosis (Annexin-V-positive and PI-positive). To increase the proportion of apoptotic and necrotic cells can be judged on the degree of influence is added to the environment of the cultivation of connections on these processes. As a positive control in our experiments we used the manufacturer's recommended connection camptothecin (Camptothecin, Sigma, USA), which have strong ability to cause apoptosis and necrosis of cells, in particular tumor.

Solutions of peptides in sterile PBS to a concentration of 1 mg/ml was prepared immediately before the experiments to study the effects of peptides on apoptosis and necrosis. To study the proliferative activity used the same solution, after which the preparation was stored in sterile tubes at 4°C.

On the day of each experiment were prepared working dilution of the peptides in the environment of the cultivation of cells. This was done with a sterile plastic 96-well plate (Corning, USA). Cultivation was prepared so that when adding 25 ál of each working dilution in experimental well cultured cells final concentration of the peptides was 400, 200, 100 and 50 ng/ml

Cultive what has been created cells

For cell culture HeLa S3 and NIH ZTZ used reagents company Invitrogen (USA) and plastic bottles, tablets, pipettes and filters Corning (USA).

Cells were maintained in DMEM prepared from the dry concentrate is dissolved in pyrogen-free distilled water (Millipore, USA). Medium containing 10% FBS, 2 mM glutamine and penicillin-streptomycin (diluted in accordance with the manufacturer's recommendations). HeLa S3 and NIH T were grown in bottles t at 37°C in an atmosphere of 5.6% CO2. Cells constantly maintained in logarithmic growth phase, which was scattering them at least 2 times a week using trypsin to remove attached cells from the surface of the culture flasks. The counting of living cells held in hemacytometer after staining aliquots of the suspension Trifanova blue.

The study of the ability of peptides to induce apoptosis and necrosis of cultured cells

Cells HeLa S3 and NIH T was removed from the surface of the culture vials were counted, and drove concentration of up to 105cells/ml and dissipated resulting suspension to 1 ml in the wells of a 24-hole culture plates.

The next day, making sure that the cells attached to the surface of the tablets, the cultural medium in all the wells was changed to fresh.

In the control wells (positive control stimulation of apoptosis and necrosis) doba is Lyali camptothecin to a final concentration of 50 μm, which was determined in preliminary experiments with cells NIH T and HeLa S3. In the control wells (negative control) was added 25 μl of fresh medium cultivation, in the experimental wells was added to 25 µl of the working dilutions of the peptides (each working dilution is 2 repeats) and camptothecin in the same final concentration. Incubation was carried out at +37°C.

24 hours after the beginning of incubation, the cells were removed from the surface of 24-well plates, transferred from each well into a separate centrifuge tube 1.5 ml, washed with 1 ml of working solution of buffer for binding dye Annexin-V FITC and besieged the cells by centrifugation (Sysmex Platelet Centrifuge RS-810). The supernatant was decanted, the cell precipitate was added 5 μl of dye Annexin-V FITC and 10 μl of dye propidium iodide (PI) and incubated for 15 min in the dark at room temperature (+25°C). Then to each tube was added 300 μl of the working solution of buffer for the binding of Annexin-V FITC and measured the intensity of green and red fluorescence.

The resulting distributions were allocated two populations - Annexin-V-positive and PI-negative cells undergoing apoptosis) and Annexin-V-positive and PI-positive cells (cells undergoing necrosis).

The histograms 13 - 28 presents the percentage of apoptotic and necrotic cells in popular is of HeLa S3 and NIH3T3 in the presence of 8 different peptides at 4 different concentrations.

From the histograms (Fig. 13-28) and Table 2 shows that the analyzed peptides in the concentration range from 50 to 400 ng/ml have a marked protective effect on cells HeLa S3 and NIH3T3, i.e. possess cytoprotective activity.

Literature

1. RF patent №2177802 (2002).

2. RF patent №2161501 (2001).

3. RF patent №2301074 (2007).

4 RF Patent №2304444 (2007).

5. RF patent №2155063 (2000).

6. Pharmactutical Research, Vol.11, No. 5, 1994, p.751-758.

7. Pharmactutical Research, Vol.7, No.8, 1990, p.787-793.

8. Synthetic peptides, sec. ed. Gregory A. Grant, Oxford Univ. Press 2002, p.283.

9. Have, Accipere, Chemical synthesis of peptides. Kiev, Naukova Dumka, 1992

10. Peptides, basic methods of formation of peptide bonds. Moscow, "Mir" 1983

Drug, possess cytoprotective activity, representing a peptide selected from the group: H-Ala-Asp-Glu-OH, H-Lys-Asp-Glu-OH, H-Asp-Glu-Pro-OH, H-Asp-Glu-Arg-OH, H-Asp-Glu-Gly-OH, H-Ala-Asp-Glu-Leu-OH, H-Asp-Glu-Leu-OH, H-Lys-Asp-Glu-Trp-NH2.



 

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