The method of obtaining n-carboxyanhydride or n - thiocarboxanilide urlencoding amino acids

 

(57) Abstract:

Purpose: in peptide chemistry in the synthesis of peptide and protein circuits due to its high reactivity, lack of undesirable side products. The inventive method of obtaining N-carboxyanhydride or N-thiocarboxanilide pretensioner amino acids total f-crystals, where R is H, R is lower alkyl, unsubstituted or substituted amino group, a hydroxy-group, a group NH-C(=NH)NH2or , benzyl or cycloalkyl, Z = O, S-lower alkyl, unsubstituted or substituted aryl. Reagent 1: N-carboxyanhydride or N-thiocarboxamide f-crystals . Reagent 2: R-O-C(=O)-X, where X is halogen. The process is conducted in an inert solvent in anhydrous conditions, in the presence of a tertiary amine, for example N-methylmorpholine, and the target product produce by using crystallization. 2 C. p. F.-ly. 1 Il.

The invention relates to methods of producing N-carboxyanhydride or N-thiocarboxanilide urlencoding amino acids, which can find application in the synthesis of peptide and protein chains.

Know the use of N-carboxyanhydrides and their derivatives as alleluya agents in the formation peptizing ties (1). These derivatives allow polucheny, pH, etc., However, can be obtained only peptides with a short chain, and the resulting products require thorough cleaning.

The purpose of the invention is a method of obtaining a new N-carboxyanhydride or N-thiocarboxanilide urlencoding amino acids, which can be used to obtain peptides with different length chains simplified by eliminating the need for activation, filtering, and mix until the formation of the peptide bond, and it also excludes racemization.

This goal is achieved opisywanym method of obtaining N-carboxyanhydride or N-thiocarboxanilide urlencoding amino acids of General formula I where R = H; R1- lower alkyl, unsubstituted or substituted amino group, a hydroxy-group, group-NHC or R1- CH2- , benzyl or cycloalkyl Z = O, S

R11- lower alkyl, unsubstituted or substituted aryl, namely, that N-carboxyanhydride or N-thiocarboxamide amino acids of General formula II: where the values of R, R1and Z above, enter into interaction with gaelicforum R11-O--X where X = halogen, R11above, in an inert diluent, in anhydrous conditions in the presence of t as a tertiary amine N-methylmorpholine, the reaction product is N-carboxyanhydride oryantasyon amino acids, using crystallization.

P R I m e R 1. Decomposition of N-carboxyanhydride depending on the base.

A. N-Carboxyanhydride valine (12 mg) was dissolved in dry distilled tetrahydrofuran (12 ml) and add triethylamine (30 μl). After the disappearance of the N-carboxyanhydride spend infrared spectroscopy.

C. N-Carboxyanhydride valine (72 mg) was dissolved in tetrahydrofuran (2 ml) and add N-methylmorpholine (25 μl). After the disappearance of the N-carboxyanhydride spend infrared spectroscopy.

The results of experiments a and b are shown on the graph presented in drawing 1.

P R I m m e R 2. N-Carboxyanhydride N-(9-fluorenylmethoxycarbonyl)-L-alanine.

A. a Mixture of L-alanine (40,3 g of 0.45 mol) and phosgene (275 ml of 3.3 M solution in tetrahydrofuran, of 0.90 mol) is stirred at a temperature 62-64aboutC for 4 h the resulting solution was left to cool to room temperature, filtered and reduced pressure to remove volatile substances. The oil obtained is dissolved in 100 ml of tetrahydrofuran, and with stirring, add 300 ml of hexane and then cooled to -20the Olya) in toluene (50 ml) is added to a mixture of N-carboxyanhydride L-alanine (8,84 g, 76,8 mmole) and 9-fluorenylmethoxycarbonyl (19.9 g, 76,8 mmole) in toluene (200 ml) at a temperature of 0aboutC. the Reaction mixture is stirred at a temperature of 0aboutC for 2 h and filtered. The volume of solvent was adjusted to 20 ml and after adding 100 ml of hexane perform crystallization, resulting 21,4 g (82% ) of crude N-carboxyanhydride 9-fluorenylmethoxycarbonyl-L-alanine. This product cleaned by grinding into powder with simple cold diisopropyl ether, after which produce recrystallization from ethyl acetate and hexane: melting point 106-107aboutWith, the infrared spectrum of (CH2CL2) 1870, 1801, 1740 cm-1; NMR spectrum (CDCl3) 6-90-7,80 (m, 8 H), 3.95 to 4,55 (m, 4 H), of 1.35 (d, j = 7 Hz, 3 H).

Calculated, % : C 67,65; N 4,48; N 4,15.

WITH19H15NO5.

Found, % : C 67,73; N 4,65; N 4,19.

P R I m e R 3. N-carboxyanhydride N-(9-fluorenylmethoxycarbonyl)-L-leucine.

A. N-Carboxyanhydride L-leucine derived from L-leucine, achieving 78% yield using the procedure described in example PA.

C. a Mixture of N-carboxyanhydride L-leucine) 9.2 grams, for 58.4 mmol) and 9-peroxyneodecanoate (15,1 g, 58,4 mmole) in toluene (125 ml) cooled amerivault at a temperature of 0aboutC for 2.5 h, filtered and the volume of solvent reduced to 20 ml Add hexane (480 ml), after which the solution is cooled to -20aboutWith during the night, resulting 18,8 g (85% ) of N-carboxyanhydride N-(9-fluorenylmethoxycarbonyl)-L-leucine. An analytical sample was obtained by recrystallization from a mixture of simple ether, methylene chloride and hexane: melting point 119-120aboutC; NMR spectrum (CCl4) 7: 35-7,91 (m, 8 H); 4.72 in (t, j = 7 Hz, 2 H); 4,58 (m, 3 H); 4,37 (t, j = 7 Hz, 1 H); is 2.05 (m, 2 H); of 1.09 (t, j = 6 Hz, 6 H).

Calculated, % : C 69,64; N To 5.58; N 3,69.

WITH22H21NO5.

Found, % : C 69,08; N 5,97; N 3,70.

P R I m e R 4. N-Carboxyanhydride N-(9-fluorenylmethoxycarbonyl)-N-8-tert-butyloxycarbonyl-L-lysine.

A. a Mixture of N-tert-butyloxycarbonyl-L-lysine (1.23 g, 5.0 mmol) and chlorotrimethylsilane (1.08 g, 10.0 mmol) in tetrahydrofuran (50 ml) cooled to 0aboutWith, and then added dropwise a solution of triethylamine (1.01 g, 10.0 mmol) in 5 ml of tetrahydrofuran. This mixture was stirred at 0aboutC for 2.5 h, filtered and added to a solution of phosgene (10 mmol) in 15 ml of tetrahydrofuran. The temperature was raised to 60aboutAnd the solution is stirred for 2.0 hours, then within what Nate was obtained 0,79 g (58% ) of N-carboxyanhydride - N - tert-butyloxycarbonyl-L-lysine. Infrared spectrum (CH2CL2) 1860, 1795, 1710 cm-1.

C. a Mixture of N-carboxyanhydride-N--tertbutyloxycarbonyl-L-lysine (0,79 g, 2,90 mmole) and 9-fluorenylmethoxycarbonyl (0.75 g, 2,90 mmole) in toluene (25 ml) cooled to 0aboutWith, and then added dropwise a solution of N-methylmorpholine (0.32 g, 3.2 mmole) in toluene (5 ml). The interaction is carried out as described in example 3, resulting in a gain 0.88 g (66% ) of N-carboxyanhydride N-(9-fluorenylmethoxycarbonyl)-N-tert-butyloxycarbonyl - L-lysine: melting point 81-85aboutWith (ethyl acetate/hexane).

An NMR spectrum (CDCl3) of 7.3 to 7.7 (m, 8 H), 4,11-4,58 (m, 5H), 2.95 and-3,20 (m, 2H); 1,90-to 1.98 (m, 2H); 0,9-1,4 (m, 13 H).

Calculated, % : C 65,57; N 6,11; N 5,67.

WITH26H30N2O7.

Found, % : C 66,33; N 6,38; N 5,67.

P R I m e R 5. N-Carboxyanhydride N-benzyloxycarbonyl-L-alanine.

A solution of N-methylmorpholine (1.06 g, 10.5 mmole) in ethyl acetate (20 ml) was added dropwise to a mixture of N-carboxyanhydride-L-alanine (from example PA) (0,81 g, 7.0 mmol) and benzylbromide (1.89 g, 10.5 mmole) in ethyl acetate (80 ml) at 0aboutC. the Reaction mixture was stirred for 1.5 h at 0aboutWith the filter is -20aboutWith, the result of which was obtained 1.20 g (71% ) of N-carboxyanhydride N-benzyloxycarbonyl-L-alanine, melting point 101-104aboutC.

An NMR spectrum (CDCl3) 7,33 (S, 5H), AT 5.27 (S, 2 H), 4,60 (q, j = 7 Hz, 1 H), of 1.61 (d, j = 7 Hz, 3 H).

Calculated, % : C 57,83; N. Of 4.45; N 5,62.

WITH12H11NO5.

Found, % : C 57,60; N 4,50; N, 5.53.

P R I m e R 6. N-Carboxyanhydride N-benzyloxycarbonyl-L-leucine.

A solution of N-methylmorpholine (0,76 g, 7,50 mmole) in ethyl acetate (10 ml) was added dropwise to a solution of N-carboxyanhydride L-leucine (0,79 g, 5.0 mmol) (from example 3A) and benzyloxycarbonylamino (1.35 g, 7,50 mmole) in ethyl acetate (50 ml) at 0aboutC. the Reaction mixture was stirred at 0aboutC for 1.25 h, filtered and reduce the volume of the solution to 5 ml Add hexane (50 ml) and then cooled to -20aboutWith, resulting in 0,89 g (61% ) of N-carboxyanhydride N-benzyloxycarbonyl-L-leucine: melting point 72-73,5aboutWith simple ether and hexane).

An NMR spectrum (CDCl3) 7,40 (S. 5 N) 5,33 (S, 2H). 4,71 (t, j = 6 Hz, 1 H), 1,80-2,04 (m, 3 H), of 0.91 (m, 6N).

Calculated, % : C 61,84; N 5,88; N 4,81.

WITH15H17NO5.

Found, % : C 61,64; N. Of 6.02; N 4,90.

P R I m e R 7. N-is 75% yield according to the procedure described in example PA.

C. Repeat the procedure described in example 5, replacing benzylchloride phenylcarbamates and N-carboxyanhydride leucine N-carboxyanhydride valine. The result was achieved in 78% yield of N-carboxyanhydride N-phenoxycarbonyl-L-valine: melting point 105-106aboutWith (chloroform and hexane).

An NMR spectrum (CDCl3): 7,30 (m, 5 H), 4,70 (d, J = 3.5 Hz, 1 H), 2,60 (m, 1 H), 1,22 (d, J = 7 Hz, 3 H), of 1.07 (d, J = 7 Hz, 3 H). of 1.07 (d, J = 7 Hz, 3 H).

Calculated, % : 59,31; N To 4.98; N, 5,32.

WITH12H13NO5.

Found, % : C 59,09; N 4,91; N 5,49.

P R I m e R 8. N-Carboxyanhydride N-ethoxycarbonyl-L-alanine.

Repeat the procedure described in example 5, replacing benzylchloride etelcharge.com. The result of this was achieved in 62% yield of N-carboxyanhydride N-ethoxycarbonyl-L-alanine: melting point 72-73,5aboutWith (ethyl acetate and hexane).

An NMR spectrum (CDCl3) 4,73 (g, I = 7 Hz, 2 H), 4,33 (g, I = 7 Hz, 1H), 1.70 to (d, I ) 7 Hz, 3 H), of 1.33 (t, J = 7 Hz, 3 H).

Calculated, % : C 44,92; N Is 4.85; N 7,49.

WITH7H9NO5.

Found, % : C 45,08; N. Of 5.03; N 7,33.

P R I m e R 9. A. N-Carboxyanhydride L-phenylalanine is produced from L-phenylalanine, achieving 53% of whenallisone (2.5 g, 13 mmol) and benzylbromide (3.4 g, 20 mmol) in ethyl acetate (130 ml) was added dropwise a solution of N-methylmorpholine (2.0 g, 20 mmol) in ethyl acetate (10 ml) at 0aboutC. the resulting mixture was stirred at a temperature of 0aboutC for 2.5 h and processed as described in example 5, the result of which was obtained 2.0 g (48% ) of N-carboxyanhydride N-benzyloxycarbonyl-L-phenylalanine: the melting point of 108-109aboutC; NMR spectrum (CDCl3) 7-35 (S, 5 H) to 7.00 (m, 5 H), 5,31 (S, 2 H), of 4.83 (m, 1 H), of 3.28 (m, 2 H).

Calculated, % : C 68,13; N. Of 5.40; N 4,42.

WITH18H17NO5.

Found, % : C 68,11; N 5,38; N 4,20.

P R I m e R 10. N-Thiocarboxamide phenoxycarbonyl-L-alanine.

A. O-Ethyl-S-methylxanthine. To a solution of ethylxanthate potassium (16.0 g, 100 mmol) in water (50 ml) added dropwise dimethylsulfate (of 12.6 g, 100 mmol) at a temperature of 4+1aboutC. After the addition the reaction mixture was washed with dichloromethane (2x40 ml), after which the combined organic fractions dried (MgSO4) and concentrate with the formation of O-ethyl-N-methylxanthine having sufficient purity; required for use in the next stage.

Century Etoxycarbonyl-L-alanine. To O-ethyl-S-methylxanthine obtained above, add races is 2,3 hours When performing a purge N2add methanol (50 ml), after which this mixture is stirred at a temperature of 45aboutEven within 0,7 hours the Reaction mixture to cool to room temperature, washed with dichloromethane (I ml), acidified to a pH of 2.5 with concentrated Hcl and extracted with ethyl acetate (2 x 50 ml). The combined organic solutions dried (MgSO4) and concentrate. Add hexane to the resulting oil is allowed to obtain 9.5 g (54% ) ethoxycarbonyl-L-alanine as a colourless solid, which melted at a temperature 74-78aboutC. This material was further purified by recrystallization from a simple and ethyl hexane: melting point 77-79aboutC; infrared spectrum (CCl4) 3397, 1716 cm-1.

C. N-Thiocarboxamide L-alanine. To a solution of ethoxycarbonyl-L-alanine (3.0 g, 17 mmol) and imidazole (1.2 g, 17 mmol) in tetrahydrofuran (20 ml) added dropwise RVG3(of 5.4 g, 20 mmol) at a temperature of 20aboutC. Stirring is continued up until a solid mass is dissolved in vysokodispersnoi suspension. This reaction mixture is then poured into a mixture of saturated solution of NaHCO3(200 ml) and ethyl acetate (150 ml) the Organic layer is separated the SUB>4) and concentrate. During maturation the resulting oil was hardened. Recrystallization of the solid substances allowed to obtain 0.75 g (34% ) N-dicarboxy - hydride-L-Allina: the melting point of 91-92aboutC; infrared spectrum (CCL4) 1750, 1695 cm-1.

D. N-Thiocarboxamide phenoxycarbonyl-L-alanine.

To a solution of N-thiocarboxanilide L-alanine (0,49 g of 3.8 mol) in 50 ml of ethyl acetate added phenylcarbamate (0.95 g, 6.1 mmole) at a temperature of 0aboutWith, and then added dropwise a solution of N-methylmorpholine (or 0.57 g, 5.6 mmole) in ethyl acetate (10 ml) at 0aboutC. the resulting mixture was stirred for 3 h at 0aboutC, filtered and concentrated with the formation of a white semi-solid substances. This semi-solid substance was dissolved in 20 ml of ethyl acetate, add hexane (150 ml), after which the mixture is cooled to -20aboutWith that allowed to obtain 0.55 g (62% ) of N-thiocarboxanilide phenoxycarbonyl-L-alanine: melting point 110-111aboutC; NMR spectrum (CDCl3) to 7.18 (m, 5H), of 4.83 (q, 1H, j = 7 Hz), 1,71 (d, 3H, J = = 7 Hz); infrared spectrum (CH2CL2) 1810, 1740 (doublet), 0,1715 (shoulder).

Calculated, % : C 52,58; N 3,61 N 5,58; S 12,76.

WITH11H9NO4S.

Found, % : C 52,75; Nna.

A. N-Carboxyanhydride O-tert-butyl-L-threonine is produced from O-tert-butyl-L-threonine with 57% yield, resulting from the use of the procedure on the basis of trimethylsilyl described in example 4A.

C. To a solution of N-carboxyanhydride O-tert-butyl-L-threonine (0,80 g, 4.0 mmole) and 9-fluorenylmethoxycarbonyl (1.0 g, 4.0 mmole) in toluene (50 ml) added dropwise a solution of N-methylmorpholine (0,49 g, 4.8 mmole) in 8 ml of toluene at a temperature of 0aboutC. the Reaction mixture was stirred for 3 h at 0aboutWith filter and remove the volatiles under reduced pressure. The residue is crystallized from a mixture of simple ether and hexane with the formation of 1.0 g (60% ) of N-(9-fluorenylmethoxycarbonyl)-0-tert-butyl-L-threonine: melting point 124-127aboutC; NMR spectrum (CDCl3) 7,08 for 7.78 (m, 8 H), 4,05-br4.61 (m, 4 H). of 1.18 (S, 9 H), of 1.16 (d, 3H, j = 7 Hz).

Calculated, % : C 68,07; N 5,95; N 3,31.

WITH24H25NO6.

Found, % : C 67,89; N 5,96; N 3,28.

P R I m e R 12. N-Carboxyanhydride ethoxycarbonyl-and-aminoadamantane acid.

A. N-Carboxyanhydride and-aminoadamantane acid get with the achievement of 67% of the output of the procedure described in example 2A.

Century Repeated the procedure, opisannuyu what was achieved in 16% yield of N-carboxyanhydride etoxycarbonyl-L-aminoadamantane acid: melting point 68-70aboutWith (chloroform and hexane), an NMR spectrum (CCl4) 4,59 (q, 2 H, j = 7 Hz), from 2.00 (S, 6 H), 1,65 (t, 3 H, I = = 7 Hz).

Calculated, % : C 47,76; N 5,51; N Of 6.96.

WITH8H11NO5.

Found, % : C 47,67; N 5,51; N 7,14.

P R I m e p 13. N-Carboxyanhydride N-tert-butyloxycarbonyl-L-alanine.

To a solution of tert-butyl alcohol (1.25 g, to 16.9 mmole) and phosgene (3.4 ml of a 5 M solution in dioxane, 17 mmol) in 80 ml of ethyl acetate are added dropwise N-methylmorpholine (3.4 g, 34 mmole) at a temperature of -50aboutC. This reaction mixture is stirred for 0.5 hours Then add N-carboxyanhydride L-alanine (0,23 g, 2.0 mmole) in ethyl acetate (10 ml), after which the mixture is stirred at a temperature of -50aboutEven within 0,75 hours Add N-methylmorpholine (1.0 g, 10 mmol) and stirring is continued for another period of 0.75 hours at the temperature of -50aboutC. the Solids are removed by filtration, the solution concentrated and after grinding to a powder with hexane obtain the target product. Recrystallization from toluene allowed to obtain 0.28 g (65% ) of N-carboxyanhydride N-tert-Butylochka - carbonyl-L-alanine: melting point 103-104,5aboutC; NMR spectrum (CDCl3) 4,71 (q, 1 H, j = 7 Hz), 1,80 (d, 3 H, j = 7 Hz), 1.70 to (S, 9 H).

Calculated, % : C 50,23; N 6,09; N 6,51.

A. N-Carboxyanhydride O-benzyl-L-serine get with achieving 68% of the output of the procedure described in example PA.

C. Repeat the procedure described in example 13 substituting N-carboxyanhydride L-alanine N-carboxyanhydride O-benzyl-L-serine, the result of which was obtained N-carboxyanhydride N (tert-butyloxycarbonyl)-O-benzyl-L-serine with 52% yield: melting point of 98-99,5aboutC.

An NMR spectrum (CCl4) 7,30 (m, 5H), with 4.64 (m, 3H, benzyl CH2and proton kernel PSA), 4.09 to (dd, 1H, j = 15,5 Hz), 3,88 (dd, 1 H, J = 15,5 Hz), of 1.65 (S, N).

Calculated, % : C 59,80; N 5,96, N 4,36.

WITH15H19NO6.

Found, % : C 59,71; N. Of 6.25; N 4,05.

P R I m e R 15. Vinyloxycarbonyl derived N-carboxyanhydride 1-amino-1-cyclohexanecarboxylic acid.

A. N-Carboxyanhydride 1-amino-1-cyclohexanecarboxylic acid derived from 1-amino-1-cyclohexanecarboxylic acid with achievement of 50% of the output of the procedure described in example PA.

C. To a solution of N-carboxyanhydride obtained in accordance with example And of 0.85 g , 5.0 mmol), and phenylcarbamate (1.2 g, 7.5 mmole) in ethyl acetate (30 ml) at a temperature of 0aboutTo add a solution of N-mstrout and concentrate. White semi-solid residue recrystallization from a simple mixture of ethyl ether, methylene chloride and hexane with the formation of 0.92 g (66% ) of N-carboxyanhydride: melting point 156,5-158aboutC; NMR spectrum (CDCl3) 7,28 (m, 5 H), 1,20-3,10 (m, 10 H).

Calculated, % : 62, 27; N 5,23; N 4,84.

WITH15H15NO5.

Found, % : C 62,03; N 5,22; N 4,77.

P R I m e R 16. N-Carboxyanhydride N-(9-fluorenylmethoxycarbonyl)-L-LEU-Qing.

A. N-Carboxyanhydride L-leucine was obtained from L-leucine, achieving 78% of the output of the procedure described in example PA.

C. a Mixture of N-carboxyanhydride L-leucine (9.2 grams, for 58.4 mmole in dry distilled ethyl acetate (150 ml) is cooled to -10aboutWith and then for 5 min added dropwise a solution of triethylamine (9 ml, 64 mmol) in 20 ml of ethyl acetate. The cooling is stopped and the reaction mixture is stirred for 15 minutes. Add a small amount of anhydrous hydrochloric acid in dioxane (4,4 M solution, 5 ml) to ensure complete neutralization of triethylamine. The reaction mixture was filtered, and the solvent is removed under vacuum. The resulting crude product is dissolved in a simple ethyl ether, filtered, and cnih careful kristallizatsii was obtained N-carboxyanhydride N-(9-fluorenyl - methyloxycarbonyl)-L-leucine with 16% yield (3.6 g). Analytical data were essentially identical to those obtained in example 3.

P R I m e R 17. L-Leucyl-L-valine.

9-Fluorenylmethoxycarbonyl-L-BA - Lin, esterified in the PIR alkoxybenzyl alcohol, substituted 2% net polystyrene (0.25 g, of 0.13 mmole, valine), placed in an apparatus for solid phase synthesis of peptides, Add dimethylformamide (5 ml) and the suspension shaken for 30 minutes Dimethylformamide removed and swollen resin was twice treated with 10% piperidine in dimethylformamide (5 ml for 5 min, and then 5 ml for 15 min) to remove 9-fluorenylmethoxycarbonyloxy protective group. The resin is washed with dimethylformamide (4 × 5 ml) and subjected to interaction with N-carboxyanhydrides 9-fluorenylmethoxycarbonyl-L-lazy-nom (145 mg, of 0.38 mmole) in dimethylformamide (6 ml) for 45 minutes Fluorenylmethoxycarbonyloxy protective group is removed as described above and the resin washed with dimethylformamide (3 x 5 ml) and methylene chloride (3 x 5 ml). The resulting dipeptide otscheplaut from the resin by treatment with methylene chloride and triperoxonane acid (6 ml, 1/1) within 45 minutes the Solution was removed and the resin washed with methylene chloride (3x5 ml) and methanol (2x5 ml). The solution connected with PR in distilled water and filtered. The aqueous solution is dried by freezing and the resulting solid is dissolved in powder together with simple ether (OH) to remove contaminants related to the resin, and dried under reduced pressure with the formation of N-leucyl-L-valine, the yield of which was > 90% . The identity of the dipeptide was confirmed by high-performance chromatography (speed of movement of the solvent front is 1.5 ml/min, the determination at a wavelength of 215 nm, 30% methanol in 0.5 M solution of chloric acid) in a joint elution known ethanol (retention time 8,49 min). It was found that the purity of this product exceeds 97% , and all pollutants have a direct link with the resin. D-leucyl-L-valine (retention time 32 min) has not been detected (detection limits < 0,1% ).

P R I m e R 18. L-Leucyl-L-valine.

Repeat the procedure described in example 16, except that N-carboxyanhydride 9-fluorenylmethoxycarbonyl-L-leucine is subjected to interaction with the free amine of L-valine on the resin using methylene chloride (5 ml) instead of dimethylformamide as solvent. The results obtained were comparable with the results obtained in example 16.

P R I m e R now in example 16, used to produce L-leucyl-L-alanyl-L-valine. After cleavage from the resin and washing with simple ether was obtained Tripeptide as a white solid, the yield of which was > 88% . Analysis by high performance liquid chromatography under the conditions described in example 16, confirmed the identity of this product, which is subjected to co-elution known standard. (Retention time 16.28 per minutes ). Drop sequence, such as L-leucyl-L-valine and L-alanyl-L-valine, were not detected (detection limits < 0,1% ).

P R I m e R 20. L-Leucyl-L-alanyl-L-valine (procedure C using tert-butyloxycarbonyl).

Tert-butyloxycarbonyl-L-valine, esterified in metilirovannye 2% net polystyrene (i.e Merrifield resin) 0.50 g, a 0.23 mmole valine), placed in an apparatus for solid phase synthesis of peptides, add methylene chloride (5 ml) and the suspension shaken for 30 minutes the Solvent is removed and the resin is treated with a mixture of methylene chloride and triperoxonane acid (6 ml, 1/1) for 30 min to remove tert-butyloxycarbonyl protective group. The resin is washed with methylene chloride (3 x 5 ml), neutralized with 10% triethylamine in methylene chloride (5 ml), PR is oxycarbonyl-L-alanine (200 mg, 1.0 mmol) in methylene chloride (5 ml) for 45 minutes, the resulting resin with the protected dipeptide washed with methylene chloride (3x5 ml). This resin again will unlock, washed, neutralized as described above. To the resin type N-carboxyanhydride tert-butyloxycarbonyl-L-leucine (240 mg, 1.0 mmol) in methylene chloride (6 ml), after which this mixture is shaken for 45 minutes the Solution was removed and the resin washed with methylene chloride (3x5 ml), methanol (3x5 ml) and methylene chloride (3x5 ml) and then dried under vacuum. Resin with tert-butyloxycarbonyl the Tripeptide is subjected to interaction with liquid hydrogen fluoride at a temperature of 0aboutC for 30 min, hydrogen Fluoride was removed and the resin is dried under vacuum. The peptide is dissolved in water, and the resin removed by filtration. The solution is dried by freezing, the result was achieved almost quantitative yield of L-leucyl-L-alanyl-L-valine. The results of the analysis performed by high-performance liquid chromatography, were comparable with the results obtained in example 18.

P R I m e R 21. L-Alanyl-L-phenylalanine (procedure with full protection).

A. To pair-toluensulfonate benzyl ester of L-phenylalanine )1.07 g, 2.5 mmole) is the mixture stirred for 0.5 h at a temperature of 0aboutWith, and then add N-carboxyanhydride N-benzyloxycarbonyl-L-alanine (0.50 g, 2.0 mmole). The reaction mixture was stirred for 2 h at 0aboutWith, then add water (20 ml) and dichloromethane (50 ml). The layers are separated and the aqueous layer washed with dichloromethane (25 ml). The combined organic fractions washed with 0.5 M Hcl solution (2 x 50 ml), 10% sodium bicarbonate (50 ml) and water (2 x 50 ml), dried (MgSO4) and concentrated. After adding hexane perform crystallization, resulting 0.66 g (72% ) of benzyl ester of N-benzyloxycarbonyl-L-alanyl-L-phenylalanine: melting point to 118.5-119aboutC.

An NMR spectrum (CDCl3) to 7.64 (S, 1 H), 6,82-7,39 (m, 6 H), 5,04 (S, 3 H) To 5.00 (S, 2 H), 4,56 To 4.92 (Sm, 2 H), of 3.07 (d, j = 6 Hz, 2H), 1,29 (d, j = 7 Hz, 3 H).

C. a Mixture of benzyl ester N-benzyloxycarbonyl-L-alanyl-L-phenylalanine (0.50 g, 1.1 mmole) and palladium coal with 10% palladium (0.1 g) in ethanol (150 ml) is shaken in the apparatus for hydrogenation Parra for 6.5 hours at a temperature of 20aboutC. the Reaction mixture was filtered, and the filtrate washed with water (100 ml). The solution is concentrated with the formation of 0.26 g (100% ) of L-alanyl-L-phenylalanine. The analysis, performed using high performance liquid chromato R I m e R 22. L-Alanyl-L-phenylalanine (procedure with partial protection).

A. To a solution of L-phenylalanine (0.33 g, 2.0 mmole) in 0.20 M solution of potassium carbonate (20 ml) and acetonitrile (30 ml) was added dropwise N-carboxyanhydride N-benzyloxycarbonyl-L-alanine (0.45 g, 1.8 mmole) in acetonitrile (6 ml) at 0aboutC. This mixture is stirred for 40 min at 0aboutC and diluted with ethyl acetate (50 ml) and 1 M hydrochloric acid (10 ml). The layers are separated and the aqueous layer was extracted with ethyl acetate (2 x 35 ml). The combined organic fractions washed with brine (30 ml), 0.5 M hydrochloric acid solution (2 x 50 ml) and water (2 x 50 ml), dried (MgSO4) and concentrate. The remainder will recrystallized from a mixture of chloroform and hexane, resulting in a gain of 0.26 g (39% ) N-benzyloxycarbonyl-L-alanyl-L-phenylalanine: melting point 121-122aboutC.

An NMR spectrum (DMSO - d6) 12,71 (S, 1H), of 8.06 (m, 1H), 7,30 (m, 5 H), FREE 5.01 (S, 2H), 4,43 (m, 1H), 4,06 (m, 1 H), 2,99 (m, 2 H), 11,19 (d, 2 H, j = 7 Hz).

C. a Mixture of N-benzyloxycarbonyl-L-alanyl-L-phenylalanine (0,208 g, 0,562 mmole) and palladium coal with 10% palladium (0.1 g) in 95% ethanol (50 ml) is shaken in the apparatus for hydrogenation Parra for 16 hours at a temperature of 20aboutC. the Reaction Is) L-alanyl - L-phenylalanine as a white solid. The analysis, performed using high performance liquid chromatography showed that the purity of the product is > 99.5% and there are no traces of racemization.

P R I m e R 23. Tert. butyloxycarbonyl-L-alanyl-L-alanyl-L-phenylalanine.

To a solution of L-alanine-L-phenylalanine (0,236 g, 1.0 mmole) in 0.20 M potassium carbonate solution (20 ml) and acetonitrile (30 ml) is added dropwise a solution of N-tert-butyloxycarbonyl-L-alanine-NSA (0,194 g, 0.9 mmole) in acetonitrile (5 ml) at 0aboutC. the Mixture is stirred for 1 h at 0aboutWith and diluted with 50 ml ethyl acetate and 10 ml of 1 N. hydrochloric acid. The phases are separated and the aqueous layer was extracted with 2 portions of ethyl acetate (35 ml the combined organic fractions washed with brine (30 ml), 0.5 M hydrochloric acid (2 x 50 ml) and water (2 x 50 ml), dried over magnesium sulfate and concentrated in vacuo. The residue is precipitated by the slow introduction of the product in chloroform cooled to the hexane under slow stirring. The resulting product is collected by filtration and dried under high vacuum, resulting in the 0.2 g (yield 49% ) of Boc-L-alanyl-L-alanyl-L-phenylalanine. Amino acid analysis indicates the presence of 1 phenylalanine and 2 alaninol. The product is a homogeneous floor-L-alanyl-L-phenylalanine.

A. BOC-L-alanyl-L-alanyl-L-phenylalanine (0.2 g, 0.5 mmole) is stirred for 45 min with 4 M solution of hydrogen chloride in dioxane (10 ml). Excess hydrogen chloride is removed by distillation in vacuum. The hydrochloride of L-alanyl - L-alanyl-L-phenylalanine get through lyophilization. This product is used without further purification.

C. To a solution of L-alanyl-L-alanyl-L-phenylalanine (all amounts obtained in the above experiment) in 0.20 M solution of potassium carbonate (20 ml) and acetonitrile (30 ml) is added dropwise a solution of N-tert-butyloxycarbonyl- (O-benzyl)-L-serine-NSA (0,194 g, 0.7 mmole) in acetonitrile (5 ml) at 0aboutC. the Mixture is stirred for 1 h at 0aboutWith and diluted with 50 ml ethyl acetate and 10 ml of 1N. of hydrochloric acid. The phases are separated and the aqueous layer was extracted with 2 portions of ethyl acetate (35 ml the combined organic fractions washed with brine (30 ml), 0.5 M hydrochloric acid (2 x 50 ml) and water (2 x 50 ml), dried over magnesium sulfate and concentrated in vacuo. The residue is precipitated by the slow introduction of the product in chloroform cooled to the hexane under slow stirring. The resulting product is collected by filtration and dried under high vacuum, resulting in the 0,202 Nile-L-alanyl-L-phenylalanine (0.2 g, at 0.31 mmole) and acetic acid (50 ml) is shaken in a device for hydrogenation according to Parr for 17 h at 20aboutC. the Reaction mixture is filtered and receive the product by lyophilization of acetic acid.

1. BOC-L-seryl-L-alanyl-L-alanyl-L-phenylalanine (all amounts previously received, without further purification) is stirred for 45 min with 4 M solution of hydrogen chloride in dioxane (10 ml). Excess hydrogen chloride is removed by distillation in vacuum. L-seryl-L-alanyl-L-alanyl-L-phenylalanine (is 0.102 g, 85% ) is obtained using high-performance liquid chromatography (RP-18, 0.1% of TN, a linear gradient of a mixture of water/acetonitrile from 95/5 to 50/50, after 1 hour) followed by lyophilization. The results of amino acid analysis is based on (S AAR-seryl-alanyl-alanyl-phenylalanine): S = 1, A = 2; F = 1. Found: S = 0,98. A = 2,03, F = 1.0 IN. Mass spectrum (M+H)+ calculated for SAAF: 395, found 395.

P R I m e R 25. Acyl peptide (65-74, VQAAItDyNG).

Acyl peptide (65-74, VQAAIDYNG) get in the reactor in the flow. Column load GEMS-glycine resin (0.8 g, 0.36 mEq/g) and balance dry dimethylformamide at a flow rate of 11 ml/min All the solvent is dried by passing through a column of molecular sieve 4. Smo is litil-DOG, F-alanine-DOG, F-alanine-DOG, F-isoleucine-DOG, F-(tert-butyl)-aspargine-DOG, F-(o-tert-butyl)-tyrosine-DOG, F-isoleucine-DOG, F(trityl)-aspargine-PSA) according to the following procedure: 1) remove protection (10% piperidine in dimethylformamide), 10 min at a rate of 11 ml/min; 2) rinsing (dimethylformamide), 5 min at a rate of 11 ml/min; 3) splitting (0.15 M U DOG in dimethylformamide), 45 min at a rate of 11 ml/min; 4) washing (dimethylformamide), 5 min at a rate of 11 ml/min; 5) repeat stages 1-4. After removing protection from terminal protective groups of the resin are removed from the column, washed with dry methylene chloride and dried in vacuum. Peptide otscheplaut from the resin by treatment for 1 h in 50% tetrahydrofuran (THF) in dry methylene chloride containing 2.5% anisole and 2.5% pentamethylbenzene. After removal of the solvents under reduced pressure and lyophilization get the crude acyl peptide (65-74) with the release of 73% .

The results of amino acid analysis is based on (VQAAIDyNG): V = 1,00, Q = = 1.00 And = 2,00, I = 2,00, D + N = 2,00, y = 1,00, G = 1,00. Found: V = 1,00, Q = 1,02; A = 2,08, I = 2,05, D + N = 2,20, y = 1,05, G = 1,00. Mass spectrum: (M+P) is based on VQAAIDyNG): 1064 found 1064.

V = valine

Q = glycine

A = alanine

I = isoleucine

D = asparagine

y = tyrosine

Op what's of Peptides in Agueons Medium VIII.

The Preparation and use of Novel L-Amino Acid N-Carboxyanhyd - The rides. J. Amer. Chem, soc. 1971. 93, No. 11 c. 2746-2774.

U.S. patent N 4267344, CL 07 D 407/02, 1981.

1. The METHOD of OBTAINING N-CARBOXYANHYDRIDE OR N-THIOCARBOXANILIDE URLENCODING AMINO acids of General formula

< / BR>
where R is H;

R- lower alkyl not substituted or is substituted by an amino group, a hydroxy-group, group

NH-C

or R-

CH2-

benzyl or cycloalkyl;

Z = O, S

R- lower alkyl not substituted or substituted by aryl,

characterized in that the N-carboxyanhydride or N-thiocarboxamide amino acids of General formula

< / BR>
where R, Rand Z have the values

enter into interaction with gaelicforum General formula R-O--X, ,

where X is halogen;

Rhas the specified values,

in an inert diluent in anhydrous conditions in the presence of a tertiary amine base.

2. The method according to p. 1, characterized in that the tertiary amine base is used n-methylmorpholin.

3. The method according to p. 1, characterized in that the n-carboxyanhydride oryantasyon amino acids, which are the reaction product, isolated by using Chris is

 

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FIELD: chemistry.

SUBSTANCE: invention concerns novel compounds of the formula I and their pharmaceutically acceptable salts. Claimed compounds display PPAR agonistic effect. In the general formula I A is O; X is a link or CH2; R1 is selected out of C1-C3alkyl; each R4 is independently selected out of group including halogen or -OC1-C3alkyl, where -OC1-C3alkyl can be optionally substituted by 1-3 F; each R5 is independently selected out of group including F, CI; R6 is selected out of group including C2-C5alkyl and -CH2cyclopropyl; m is 0; n is integer 1-2; p is integer 0-1.

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FIELD: chemistry.

SUBSTANCE: invention relates to a method of crystallising N-carbonic anhydride of glutamic acid benzyl ether. The disclosed method includes dissolving N-carbonic anhydride of glutamic acid benzyl ether in a solvent heated to a temperature of 40°C or higher but lower than the boiling point thereof, and the amount of which is 0.5 l or more per mole of N-carbonic anhydride of glutamic acid benzyl ether. A weak solvent is then added in amount of 1.4 l or more per mole of N-carbonic anhydride of glutamic acid benzyl ether at a temperature equal to higher than 40°C, but lower than the boiling point thereof. Further, crystals are precipitated at a temperature equal to higher than 40°C, but lower than the boiling point, and the crystals are cooled to obtain crystalline polymorphs of N-carbonic anhydride of glutamic acid benzyl ether. The solvent which is heated to a temperature of 40°C or higher, but below the boiling point, is ethyl acetate and the weak solvent is an aliphatic hydrocarbon. The invention also relates to crystalline polymorphs of N-carbonic anhydride of glutamic acid benzyl ether obtained using said method.

EFFECT: present method enables to obtain crystalline polymorphs of N-carbonic anhydride of glutamic acid benzyl ether, having high bulk density and excellent storage stability.

5 cl, 5 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula 1 where R is hydrogen or a C1-4-alkyl group; R1 means a group selected from the group consisting of structures represented by formulas (Ia), where R2 means hydrogen or a C1-4-alkyl group; R3 means hydrogen, a halogen, CF3, CN or C1-4-alkyl and R4 means hydrogen, a halogen or a C1-4-alkyl; a=0, 1, or 2; b=0, 1, 2 or 3; C=1, 2 or 3, and Ra, Rb, Rc and Rd represent, independently of one another, H or a C1-4-alkyl; X means a C2 aliphatic hydrocarbon bridge optionally containing a double bond or a triple bond or a hetero-atom selected from O and S, or CH(CH2)CH-; Y means hydrogen, a halogen, a C1-4-alkyl, C1-4-alcoxy or C1-4-hydroxyalkyl; Z means a C1-4-aliphatic hydrocarbon bridge optionally containing one double bond and/or one hetero-atom selected from O, S, N and N(CH3), or means a C2-4-aliphatic hydrocarbon bridge fused with a C3-6-cycloalkyl, optionally containing one or more double bonds or with a phenyl ring, or means a C1-4-aliphatic hydrocarbon bridge substituted with a spiro-C3-6-cycloalkyl, optionally containing one or more double bonds; or its pharmaceutically acceptable salt, or a stereoisomer, or a pharmaceutically acceptable salt of the stereoisomer. The compounds according to the invention are obtained due to reductive amination of the benzaldehyde of formula 4 by means of a primary amine R1-NH2, the introduction in the interaction of the obtained secondary amine of formula 2 with ether formyl - or oxocyclohexanecarboxylic acid of formula 3, where X, Y, Z, R1, R, Ra, Rb, Rc, Rd, b and c have the meanings as specified for formula 1, and R' means -CHO or =O, and, optionally, the hydrolysis of the obtained ester of formula 1. The invention also relates to an intermediate compound of formula 2 or a salt thereof. The compound of formula 1 according to the invention is intended for use in the preventive and / or therapeutic treatment of a disease or disorder mediated by CXCR3 receptor as a medicament or as part of a pharmaceutical composition.

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20 cl, 6 tbl, 160 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to (i) essentially crystalline melagatran in the form of hydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 21.1, 10.5, 7.6, 7,0, 6.7, 6.4, 6.2, 5.7, 5.4, 5.3, 5.22, 5,19, 5.07, 4.90, 4.75, 4,68, 4.35, 4.19, 4.00, 3.94, 3.85, 3.81, 3.73, 3.70, 3.63, 3.52, 3.39, 3.27, 3,23, 3.12, 3.09, 3.06, 2.75, 2.38, and 2.35 Å and/or water content 4.3%; and (ii) essentially crystalline melagatran in the form of anhydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 17.8, 8.9, 8.1, 7.5, 6.9, 6.3, 5.9, 5.6, 5.5, 5.4, 5.3, 5.2, 5.0, 4.71, 4.43, 4.38, 4.33, 4.14, 4.12, 4.05, 3.91, 3.73, 3.61, 3.58, 3.56, 3.47, 3.40, 3.36, 3,28, 3.24, 3.17, 3.09, 3.01, 2.96, 2.83, 2.54, 2.49, 2.41, 2.38, and 2.35 Å. Invention also relates to a method for preparation of indicated form, a method for interconversion of anhydrite form, to use of indicated compounds as pharmaceutical agent, and to preparation of drugs. Pharmaceutical preparation is suitable for treatment of condition, in case of which inhibition of thrombin is needed or desirable. Invention provides a method for treatment of such condition.

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14 cl, 4 dwg, 3 tbl, 9 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: biotechnology, medicine, proteins.

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EFFECT: valuable medicinal properties of polypeptide.

19 cl, 33 dwg, 1 ex

FIELD: biotechnology, medicine, infectious diseases, medicinal microbiology.

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EFFECT: valuable medicinal properties of composition.

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Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention represents group of Neisseria proteins eliciting antigen properties. Protein eliciting antigen properties comprises fragment of protein ORF 40 (amino acid sequence of ORF 40 is given in the invention claim) that involves 7 or more conservative amino acids arranging in succession. Proteins as components of proteins in the claimed group are used as a medicinal agent or for it manufacturing for treatment and prophylaxis of infection caused by Neisseria, and for manufacturing the diagnostic preparation. Invention relates also nucleic acid encoding the Neisseria protein. Nucleic acid is used as the protein. Applying the invention provides the maximal recognition and reactivity between strains of Neisseria. Invention can be used in manufacturing curative-prophylactic preparations with respect to Neisseria meningitides.

EFFECT: valuable biological and medicinal properties of antigen.

27 cl, 51 dwg, 10 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

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EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

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