SU784218A1 - Synthetic analog of natural taphcinpeptide possessing phagocytostimulating activity - Google Patents

Description

.one .-,

The invention relates to the field of synthetic peptides, namely to. A new analogue of the Natural peptide taftsin formula

GC-i-Qen-PVO-Av lg -CH COOH-WaO,.

able to stimulate phagocytosis. This analogue of tuftsin can be used in medicine, since a number of diseases (sickle-shaped anemia, myelofibroe and 4piyfrHe) are associated with a decrease in phagocytic acid. leukocyte tI.

It is possible to use ta: fcina instead of -globulin in the treatment of infectious diseases: after injuries of the spleen, which are exposed to a sharp decrease in the body size, taftsina MSzzl. :; At:

Numerous synthetic analogs of tafcine, which are formed by modifying one or two amino acid ostagcs of the molecules 111 and 1; 4}, are currently known. These analogues are. In general, they have a lower activity compared to tufcine. .

The purpose of the invention is to expand the arsenal of means of influence; alive,

 . . 2

organism, the creation of analogue with the same biological activity as taftsin, but more nisbcToro and economical than taftsin ..

. This is achieved by describing the synthetic analogue of the natural Peptide:

...: Gitvj-Gen-Pro- V-g-CHi.COOH iHjO,

 possessing phagocytosis-stimulating activity. This analogue, C1 p 1 -tafcin, is, like, mutafcin itself. fragment domain sc t. Human chains of human immunoglobulin ii class TgG with amino acid sequence 341-344 15.

..Yri determination of phagocytic ak. , Tivnost Segmen about Nuclear Neutrophils

 rats according to the method B.C. Gosteva 2 & et al., it was found that the cochlea; E1Inenie activates phagocytosis to the same extent as tuftcin itself (Table 1

;. and 2)-. -:.

According to this technique for experiments

25 take rat blood, diluted with 2% sodium limonate, that ratio 1: 1 and. studied compounds. AT . izoto is used in control experiments;: sodium chloride solution. Do3 (). The daily culture of coagula-positive pathogeynog1E of the strain STaphy i osossis: aureus at a concentration of 1 billion in 1 ml. The mixture is incubated in a teaper at 37 ° C for 30 minutes. Samples are prepared for the procedure, fixed with a Metromor according to Romayovsky-Gyms, 100 segmented nuclear neutrophils are counted, the number of absorbed bacteria is determined from them (% of active phagocytes) and the average number of ba phterocytic number. Kteriy in one segmented sod neutrophi jie. Phagocytic indicators are given in Table. one, . . . .. ,, The advantage of the described compound is: the use of more expensive Biax and the available amino acids as compared with tufcine (for example, replacement; threonine by more than 50 times less: glycemia). The fcty synthesis process, -Sep 3-tafdine, is less labor intensive, as it does not require the introduction of additional. protective groups for the hydroxyl function of threonine, guanidine, arginine function, etc. which simplifies the cost of its possible industrial synthesis. ... . Cciy, GEn J-taftsyn is synthesized by the well-known method S and 4} steps of TEMPARATION of the peptide Target from the C-terminus according to the above scheme. I protect the guanidine group of arginine. (By the formation of internal salt, by which it is possible to protect its carboxyl functions. Amino acids combine to form benzyl Loxycarbonyl- or tert-buloxycarbonyl derivatives of active ethers of ethers. Deprotection in the stages of dipeptide and tripe isopropionic acid. / ice СН С UN accordingly. The product is purified at the stage of a pure tetuptigtide by xrsematography on a silica gel. After removing the hydroxy groups by hydrogenation, a pure product is obtained over palladium .. for synthesis, a new compound nor {1-glycine, 2-glutamine) -tafcina using amino acids and their derivatives firms Reanal, Vengri. All amino acids, except glycine, on 1-configuration. Melting temperatures determined in open capillaries are given without corrections. The homogeneity of the compounds obtained is checked on Eastman plates. Chromatography, R-f- mobility. the following systems: A - Ezenzene etylacetate - acetic acid (30:10: 0.5); B - n.butanol - ethanol - water - acetic acid (80: 10: 30: 5); B-th-butanol-pyridine-water-acetic acid (30: 20: 24: 6); Mr. N. Butanol - water - acetic acid (4: 1: 1), as well as electrophoretic j. Odvizhnos-by reference to the hist di. . ; Synthesis scheme (1-glycine, 2-glutamin (α-Tfine on FN-15 paper in 1N. (PH 2.4) and 5N. (PH 1.4) with acetic acid. The angles of rotation were determined using Perk in Elmer 141 polarimeter For all compounds, the elemental analysis data satisfactorily coincide with the calculated content of C, N, N. The structure of the peptide is confirmed by amino acid analysis, which is performed with a BiocaE-200 analyzer after hydride of the peptide in a sealed ampule at 24 hours. Example of synthesis. Benzyloxycarbonyl- propnyl-arginine (1). iC 4.00 g (11 mmol) of benzyloxycarbonyl-prolium p-nitrophenyl ester 70 ml of dioxane with stirring add 1.57 g (9 mmol) of arginine in 40 MP of water. The resulting solution is transferred 2, then evaporated in vacuum () to dryness and the residue is dissolved in 15 mL of dimethylformamide. The solution is stirred for another 24 hours, then added 10.0 ml of ethyl acetate and allowed to stand in the refrigerator for 6 hours. The precipitate is filtered off and washed with ethyl acetate. After recrystallization from absolute salt and ether, the yield of the pure product is 3.47 g (-95%), mp. 137-140C, -46.6 (C1, NgO); R 0.00 (A), 0.53 (B), 0.66 (C), 0.65 (D), 0.5-6 (pH 2.4). tert-Butyoxycarbonyl-glutamyl-prolyl-arginine (3). 3.35 g (8.3 mmol) of benzyloxycarbonyl-prolyl-arginine (1 is dissolved in 10 MP of glacial acetic acid and 10 ml of 4N HBg / ice is added. After 1 h, the mixture is evaporated to dryness in vacuo () and the resulting oil The mixture is triturated under refined solid ether, dried at 1 mm Hg above caustic, then dissolved in 100 ml of water and treated with AB 17x8 resin (15 ml) to obtain free length - argyine ( 2) A solution of 4.05; g (11 mmol) of tert-butyloxycarbonyl-glutamine p-nitrofenide ester was added to a solution in 20 ml of water with stirring 45 ml of dioxane.

After 2 h, the solvent is evaporated in vacuo () and the residue is diluted with 20 Mrt of dimethylformamide, the solution is stirred for 48 h, 200 ml of ethyl acetate are added to it and the solution is kept in S fridge for 24 hours. The precipitated precipitate is filtered, washed with ethyl acetate and ether. Exit 3.74 g (79%), amorphous., ki. The precipitated precipitate is washed out, washed with ethyl acetate and ether. The yield is 3.74 g (79%); amorphous, cijS-64, 7 (C1, HgO); Kg 0.00 (A)

0.37 (B), 0.58 (C), 0.59 (D), Esch 0.54 (pH 2.4). ,

Benzyloxycarbonyl-glycine pentafluorophenyl ether (4).

To a solution of 6.30 g (36 mmol) of benzyloxycarbonyl-glycine in 160 ml of ethyl acetate was added with stirring 6.60 g (36 mmol) of pentafluorophenol in 30 ml of ethyl acetate. The solution is cooled before and 7.40 g (36 mmol) of dicyclohexylcarbodimide in 30 ml of ethyl acetate is added. The reaction mixture was held for 1 h at 10 h., Then at h at room temperature, dicyclogol eksilkarbamid was filtered off, evaporated from ethyl acetate until 200 ml of hexane was added and the mixture was heated in a refrigerator for 72 hours. The precipitated crystals were filtered off, washed with hexane. Yield 10.30 g (9: 2%); m.p. Sl-ea C, Rf 0.89 (A), 0.88 (B), 0.78 (B), 0.89 (D). . .

Benzyloxycarbonyl-glycyl-glutamyl-prolyl-arginine (6).

3.00 g (5.7 mmol) of tert-butyloxycarbonylglutamyl-prolyl-arginine (3) is dissolved in 20 ml of glacial acetic acid and 20 ml (20 mmol) of 1N is added. . СHjCOOH ,. incubated at room temperature for 1 hour, evaporated in vacuo () dosuphagocytic display of spruce leukocytes after exposure to tufcin

Xa, the resulting oil is triturated under 200 ml of ether to form a solid, kept for 2 hours at 1 mm Hg. over caustic potash. It is then dissolved in 100 ml of water and treated with AB 17 X 8 ion exchange resin (15 ml) to obtain free glutamyl-Prilyl-arginine (5). To a solution of the tripeptide (5) in 6 ml of water, with stirring, 2.50 g (6.2 mmol) of benzyloxncarbonyl-glycine (4) pentafluorophenyl ether in 4 MP dioxane are added. Withstand solution

2 h at room temperature, evaporate the solvent in vacuo () to dryness — and bcTaTok is dissolved in 8 ml

5 dimethylfrpmcea. The resulting solution is stirred for 48 hours, 200 MP of ethyl acetate is added, the precipitate formed is filtered off, washed with 80 ml of ethyl acetate and 10 ml of ether. The course of 2.94 g (87%) is used. The substance is purified on silikagel (column 30x600) in system B. The yield of the pure product is 1.71 g (51%); Ect 3% ° -63 (C1, 5% SNEON);

, Ri 0.001 (A). 0.35 (B), 0, 4 (B), 5, 0, 5b-1G), En | 5 0.50 (pH 2.4).

(1-glycine, 2-glutamine) taftsin (7). 6;, 66 g (1.1 mmol) of benzyloxycarbonyl-glycyl-glutamyl-prolyl-arginine (6) is hydrogenated at atmospheric pressure in the presence of palladium black in a mixture of 20 ml of methaol and 0.1 ml of acetic acid for 32 hours. , rubbed under ether. Dried | over phosphorus pentoxide at 1 mm Hg the product is electrophoretic and chromate graphically homogeneous. The output of 0.45 g (86%), so pl. (with different); ) -77 (C 0.6, 5% CHjCOOH); R "0.00 (A), 0.08 (B), 0.18 (c), 0.15 (D), YO" 0.92 (pH 1.4). T and l and c and 1 Blood (segmentoderny neutrophils) and (1-glycine, 2-glutamine-taftsina)

% Active 76.0 -81.2 phagocytes

Phagocytic

5.4

number4.6

7.0 9.0 7.7 6.8

4.9

8.9 7.1 82.6 66.0 81.3 86.4 86.0 73.0

 -. -. . .,. -, -. m. and 6l and c 2

Investigations of the phycocyte AUSY-activity of segmento-nuclear leukocytes (paracetes) of the bwy rat when exposed to the diethaph of the tafcin analog - Gty-GIn-Pro-Arg f eHgCOOH-ZN.O

1.54 2.29 2.05

2.23 33.6 32.8 il p and m e h a n and e: -.:

Claims (5)

1.Cunningham A., Ru t i shauser U CaP W., Gottlieb P.O. , Wax da 1 M.J Ed “lman G.M. The covalent structure of a Human TG-Jmmunogtobulin. Vii. Ami no Acid Sequence of Heavy Chains Cyanogen Bromide Fragments H. -H. Biochemistry, 1970, v. 9, No. 16, p. 3161-3170. 2.Edelma.n G.M. The covalent structure of Human -jr Gmmunog} o .buHn. Xi. l unctional PtrP cat-ion. B (ocheftii stry, 1970, v. 9, no. 16, e. 3197-3205 .. 1.91 2.45 2.21 29.7 39.3 38.2 3.Wajjap V.A. Molecular Basis of. Familiai and Acgulred Phagocytosis Deficiency Jhvolvlng the fetrapeptide, Thr- -Pro-Arg, Tuftcin. - Expl. Cell.elol, 1978, V. b, No. 1-2, p.ll4126. . 4.Koriopin & ka D. / Mawrocka E., S i emi on J.Z., SIbpek S., Szymam i ec S t Kionowcka E., Partial seguences of histones With tuftsin actiVity. 3nt. a.Peptide Protei n Res. 1977, V. 9, f 1, p. 71-77. 5.Const ntopouiOS A., Najjar V.A. Smith J.W. Tuftcin deficiency: A new syndrome with defective Phagocytosis .. J. Pediat., 1972, v. 80, 4, p.564572. . . .... -. , 6v Gostev B.C., Peter Shina.M.H., P6povkina S.A., A.K. Phagocytosis, kits and blood coagulation with a lack of protein: in food In the book. Nutrition issues. Proceedings of the Academy of Medical Sciences of the USSR, v.13, no. 2, M., 1950, p. 110-116. A is the number of bacteria in a segmented turf neutrophil when exposed to drugs; Aj is the number of bacteria in a single segmented turf neu rophile in the control; . : P is the number of active phagocytes when exposed to prepyrate; ; , -; -. .., .., /. . . Rc is the number of active phagocytes in the control.