SU767090A1 - Method of eliminating sulfenyl groups from n-sulfenyl-aminoacids and n-sulfenylpeptides - Google Patents

Description

(54) METHOD OF SEPARATION OF SULPHENYL GROUPS FROM N-SULPHIVYLIAMYLKYLOTS AND N-SULPHENYL-PYTOPIDES

-. :: -:--:/:, ,- one , ; /; The invention relates to improved cleavage of: the sulfenyl protective groups in M-sulfenylMyno-acids and N-sulfenylpeptine and can; to find an application in the synthesis of peptides. . . . In peptide chemistry, o-nitrophenylsulfenyl (NFS), 2,4-dinktrofenylsulfenyl and pentachlorophenyl group | 1 are used as peptide chemistry, but the Nfse-1CiTya group is most prevalent. , In the process of cleavage of the NFS group, Kylslt mi (or other compounds with an acidic character) in P1yptydy containing residues; Methylcysteine, S-tritylcysteine, tryptophan, and tyrosine are observed, and side reactions are observed associated with the action of these residues 5b with the o-nitrofenylsulfenylLI cation. For example, in the presence of the fatty group of M-Nfs-5-acetaminomethylcyne thein chloride with hydrogen, instead of the expected S-acetaminomethylcyneTine, S-Nfs-cyTein 12 is obtained}. To avoid this, nucleolyl reagents are currently used to cleave the sulfenyl groups, which cleave the sulfenyl amide bond without forming the sulfenyl core. Thioamides are used for the prepaE of the application. For example thio. Acetamid, tyobejamid. The reaction is carried out in an organic solvent in the presence of an acid. H $ 1 1 mole NSF-pepTiya or NSF-amino acid (containing a H c-group) using 2 trOf 3 or 4, 2} mole tyoatsetamida i NylosTaTkstl known method is ysybleovanie large izbytkatiodtsetamida that hinders vscheLenie and purifying the deprotected peptide or amino acid prsLo Mercury Nfs-G1 groups. The purpose of the inventive invention is to simplify the process of removing Nfs protective f-pynri. This goal is achieved by describing the NttJM method by cleaving the sulfenyl groups from the M-sulfenylamino acids and H-bisphenyl peptides by acting on the thiol-protected functions of the M-sulfenylamino acids N-Ulphenyl peptides with thioamide in an organic solvent in the presence of an acid, which consists of: that the sulPhenyl group is quantitatively cleaved by the action of 0.5-0.6 mothioamide per 1 mol of the M-sulfyl derivative. A preferred variant of method 6a is to use the o-nitrophenylsulfenyl group as the thioamide thioacetamide and thiobenzamide as the sulfenyl group, protection of thiol functions use the S-trityl group. The reaction is carried out in an inert solvent or mixture of inert solvents in the presence of a weak acid1 (preferably acetic acid) for the protonation of the aminopeptide, for example .MQf), ethanol is acetic acid, and this NOL is ethylacetate-acetic acid The temperature of the reaction can / can be ranging from 0 to 40 ° C, but room temperature is preferred, the reaction time is 0.5-24 hours, usually 2-3 hours. Treatment of the reaction mixture after cleavage of the NSF group is carried out according to standard methods of synthesis. The method allows to reduce the consumption of thioamide by more than two times (4-8 times) in comparison with the known cleavage method of cleavage - HFC-group-amyamide 1 and. Saving reagent. It facilitates the elimination of the target product and is of particular importance. when splitting off the Ifs group and NFS-amino acids .. after them. separation into enantiomers by different .me.ds (upon receipt of the most active amino amines from racemates). . As an example of the use of thioamide (thiobenzamide). In an amount of 0.5–0.6 mol per 1 mol of the NFS compound, a procedure is presented for the synthesis of desaminooxitocin, a highly active anthrax peptide hormone, oxytoHcc-TyrHflg - Ofte. Dtsgk, Honip .. (c) Hre- Ttip- jje-one ii) -f muoSeffjaHiff 2) (trt) - omp (I Mpr (Tpmj - Tcr - Me - O fie (Sj Hnp {Tpmj - Tup-Hftt -OH Hcpf- G f I

O) Hnp (Tpff) -Tup-Hflt-rnH AcH-T- Cys (Gy / n) - Prv-Leu-Rot-NHi

 -f iodine / 80% ijKcycHon Kttejtoma (nj Appr - Tup - ple - rjJH- AcH - qie - Pro - Lei - lead

 0.5-0.6 mol of thiobenzamide was consumed per mole of the NSF peptide. W qing. All intermediates obtained after the cleavage of the NFS group can also be used for the synthesis of oxytrcin. or its other analogues. As the S-protection, 5 trityl groups are used, however, S-acylaminomethyl groups can also be used, Desamino-Oxytocin is synthesized from two blocks, the N-terminal tripeptide and the C-terminal hexapeptide (3 + 6), which are obtained by sufficiently increasing the peptide chain using pentafluorophenyl (Opf) esters of NFS-amino acids and Opf-ester of S-trityl.-2-mercaptopropionic acid. Compounds 1-10 (cf. scheme), Opf-esters, obtained from the corresponding acids, dicyclohexylcarbodiimide (DCGC) and pentafluorophenol, can be used to form a peptide bond also without their isolation in an individual form, as in this example in the case of L- derivatives glutamine, L-tyrosine and..M-terminal tripept.ida. Activated tyrosine esters with a free hydroxyl group are prone to intermolecular condensation, a. activated glutamine esters undergo intramolecular cyclization with the formation of glutarimide derivatives. -. In order to accelerate the completion of the peptide bond formation reaction (aminolysis of pentafluorophenyl ethers), 0.5-0.7 equivalents of a tertiary base (N-methylmppholine) is added to the reaction mixture, which partially neutralizes the release of acidic pentafluorophenol in the reaction. At the last stage of the synthesis, the diluted solution of ditrityldeaminamine is treated with iodine and Proam-fJeu-Gly-NH2 I -t-MiDC Desamino-oxytocin Pro-fJeu-Gly-NH2-Cd (h) C - (Tpmj - Pro-leu-Gmi - NHz (z) Jl) is prepared. -h muoSsHSc iuS g) + - Hipc AcH-0 Hp - Cys (t / t) - fjfo - Lei - Gly - NHj, / JI il} thiobenzene d) l-H10C-Gy-OP1p ") -cis (grt ) npo-Jleu-mu-NHg sj -t-muoffHifffrud Oflip

The following abbreviations are used:

NFS - o-nitrophenylsulfenyl, 2-nitrophenylsulfenyl, o-nitrophenylthi o; (Trt) - S-TRITIA; OPF - pentafluorophenyl ether; NOPF - pentafluorophenol; DCC - dicyclohexylcarbodiimide; ACH-, Gly-, -Gl-, -Ile, -Ley-, -Pro-, -Tir-, -Cis-, Mpr - respectively L-asparaginyl, tlicyl, 1-glut1-minil, L-isoleucil, L-leucil , L-prolyl, L-tyrosyl, L-cysteinyl, 2-mercaptopropionyl. Synthesized deaminoxytitcin was tested for optical purity by analyzing volatile derivatives (N-trifluoroacetylisopropyl ethers) of the amino acids of the deamino-oxytocine hydrolyzate by gas-liquid chromatography using optically active stationary phase (H-trifluoroacetyl cyclohexyl ester H-trifluoroacetyl-i-3-ch-3-chyro 3-ch-3-chyrometra 3). did not exceed 0.5%, including isoleucine derivative.

When characterizing the products, the melting points were determined in open capillaries without correction, the purity of the products was checked using thin-layer chromatography on silica gel (Silufol, KavaEieg, Czechoslovakia plates), using the following chromatographic systems:

(1) benzene,

(2) ether

(3) ethyl acetate,

(4) chloroform + methanol + acetic acid 85 + 10 + 5 (ml),

(5) n-butanol + acetic acid + water + 4 + 1 + 1 (by volume).

For semi-preparative chromatography (for purification of substances for elemental analysis), silica gel is also used. Used in the synthesis is 0.1 M sulfuric acid solution, 0.2 M sodium hydrogen carbonate solution, 2 M sodium carbonate solution are aqueous solutions.

Example 1. Pentafluorophenyl ether o-nitrophenylsulfenyl-5-trityl-1-cysteinag NFS-Cys (Trt) -Opf (1).

To a solution of 12.1 g (23.4 mol mol, mm) of o-nitrofeylsulfenyl-5-trityl-1-cysteine in 30 ml of dioxane cooled to 15c is added a mixture consisting of 8.2 ml of a ZM solution of DCC in dioxane and 8.2 ml of 3 M solution of pentafluoroello in dioxane. The reaction mixture is stirred for 3 h, the precipitated precipitate is dicyclohexylcarbamide is filtered off and washed on the filter with dioxane (ml). The filtrate is evaporated to an oily residue, which is mixed with petroleum ether (mm). The solvent is separated de. cantation after the last wash

the residue is further dried under vacuum (40 ° C, 0.1 fvjMHg). The yield is over. hardened pentafluorophenyl ether (T) 15.1. g (90%). mp 70-72 ° C; Rf 0.75 (1) ..

A small portion (0.5 g) of pentafluoro phenyl ether (I) was dissolved in α-benzene (2.5 ppm) and the solution was applied to a column (cm) filled with silica gel and eluted with benzene. Collect the first fraction, painted in

About yellow color, the solvent is evaporated (40 ° C) in vacuo, the residue is triturated with petroleum ether (ml) and dried in vacuum over phosphorus pentoxide and sodium hydroxide. Output

5 NFS-Cis (Trt) -OPF in the process of chromatography 80%; m.p. 77-79 ° C; o (3 - 12.0 (s 0.5; dioxane).

Found,%: C 60.10; H 2.95; N 4.02.

0Cj. H2aP5N204Ss (682.69).

Calculated: C 59,82; H 3.40;

N 4,10.

o-Nitrophenylsulphenyl-5-trityl-1-cysteinyl-L-prolyl-I-leucylglycylamide, Nfs-Cys (Urt) -Pro-Leu-Gly-MH2 (2).

five

To a solution of 14.0 g (20.5 mm) of Nfs-Cys (Trt) -Opf (1) in 40 ml of dioxane, cooled down to about 15 ° C, add an extract of 5.8 g (20.5 mm) 1 -prolyl-1-leucylglycylamide in 20 ml of dimethylAorm0 amide. The mixture was stirred for 0.5 h, then 1.6 ml (70%) of N-methylmorpholine was added dropwise and left overnight at room temperature.

The solvents are evaporated in vacuo,

5 residue is triturated with ethyl acetate (100 ml). The ethyl acetate solution was washed successively (3x30 ml) with an OD M solution, sulfuric acid, water, 0.2 M sodium hydrogen carbonate solution, water, dried over sodium sulfate and evaporated (40 ° C) with ethyl acetate in vacuo. The remaining oil is stirred (35 ° C) with petroleum ether (Mjri).

The solvent is separated by decantation, and after the last wash, the residue is dried in vacuo. The yield of yellow tetrapeptide (2) is 14.4 g (90%); mp. 120-122 ° C; tc 3 - 62.2 (s 1.0; diokean); R 0.55 (4), 0.86 (5).

 Found,%: C 62.54; H 5.74;

N 10.26.

% “46 60652 (782.99). Calculated,%: C, 62.89; H 5.92;

M 10.73.

5 o-Nitrophenylsulfenyl-1-asparaginyl-5-trityl-1-cysteinyl-1-prolyl-1-leucylglycylamide, NFS-Cys (Trt) -Pro-Leu-Gly-NHrj. (3).

To a solution of 14.3 g (18.3 mm) of Nfc0-Cys (Trt) -Pro-Leu-Gly-HH, (2) in 60 ml of ethanol was added 1.4 g of Guyo mm) thiobenzamide and after dissolving reagent 3. ml of acetic acid. The mixture is kept at room temperature for 5–3 h, filtered, and the precipitated by-product precipitates a 767090 LOW. The filtrate is evaporated in vacuo, the residue is dissolved in a mixture of 20 ml of ether and 100 ml of an OD M solution of sulfuric acid. The ether layer is separated, the aqueous layer containing the aminotetrapeptide sulfate is extracted with ether to the colorless ether layer (ml) and 2M is added at 5 ° C, sodium carbonate solution until the pH of the solution is 9.0. The precipitated aminotetrapeptide oil is separated and washed with water (ml). The water layer is additionally extracted (3x20 ml) with a mixture consisting of chloroform and ethanol 10 + 1 (by volume), which is added to the aminotetrapeptide oil. After evaporation (35 ° C) of organic solvents to the solid residue {11 g 16, 5 mm, 90%) of the aminotetrapeptide are added 50 ml of dioxane, 7.5 g (16.5 mm) of O-nitrophenylsulphenyl-1-asparagine pentafluorophenyl ester and, after dissolving the components, 0.9 ml (8 mm) is added (in methyl morpholine. The mixture is stirred for 5 hours, the solvents are evaporated (40 ° C). The residue is dissolved in 100 ml of ethyl acetate and the solution is successively washed (ml) with 0.1 M solution of acid .. with water, 0.2 M solution of sodium hydrogene carbonate, water, dried over sodium sulfate and ethyl acetate (40c) evaporated in vacuo. After trituration with ether (2x30 ml yield of pentapeptide ( 3) 13.9 g (90% of the calculation for the aminotetrapeptide; 85% based on the protected tetrapeptide, mp 133-134 ° C; Rr 0, io (4), 0.60 (5). A small part. ( 0.3 g) protected Pentapeptide (3) is dissolved in in ethano-le (3 ml) and the solution is applied on a head (C 12 cm) filled with silica gel Lem. Elute with ethanol, the first fraction, O1, which is yellow, is discarded; collect the second, the main yellow colored fraction, evaporated (40 ° C) solvent and the residue grow. live with ether (ml). The yield of purified protected Pentapeptide 85%; m.p. 137-139 ° C; t5 62.9 s 0 dioxane). Found,%: C 59.85; H 5.61; N 12.20; (897.09). c gHgriN-iOeS i Calculated,%: C 60.25; H 5.84; N 12.49. 2. About Nitrophenylsul Example of phenyl-1.-glutaminyl-1-asparaginyl-5-trityl-L-cysteinyl-L-prolyl-L-leucylglycylamide, Nfs-Gln-Asn-Cys (trt) -Pro-Leu-Gly-ML (five). 6.4 g (21.4 mm) of o-nitrophenylsulphenyl-1-glutamine are suspended in 30 ML of dioxane, the suspension is cooled to 15 ° C, and a mixture of 7.4 ml (22.4 lm) is added to it 3 M of solution of clohexylcarbodiimide, and in ioxane and 21.4 ml (64.0 mm) of M of pentafluorofecol solution in dioxane. The reaction mixture is stirred at 5 ° C for 6 h, and the yellow precipitate of o-nyrophenylsulphenyl-1-glutamine dissolves and the dicycloheca L-urea precipitates, which is filtered, washed on the filter with dioxane (ml) and the filtrate is evaporated in vacuo to an oily residue. ether (50 ml) was added to the residue, and a little soluble precipitate (1.0 g) was filtered off. The elementary siege analysis data are consistent with the assumption that the cage is an o-nitrophenylsulfonyl-1-glutamic acid imide. Found,%: With 47.48; H 4.09; N 14.33. Calculated for imide with o-nitrophenylsulfonyl-1-glutamic acid. C, N.M.OdZ (281.29)%: C, 46,97; H 3.94; N 14.94. , Calculated for Nfs GLN-OP H-toFgNVjOsS (465.36),%: 1; H 2.60; N 9.03; AO, m.p. 159С (decomposed); Sun 0.35 (2) 0.70 (3). According to IR spectroscopy, the nitrile group is absent. The ether solution is evaporated to low volume and petroleum ether (80 ml) is added. The precipitated oil is triturated with petroleum ether (2x30 ml). The solvent is removed by draining, and after the second wash by evaporation (35 ° C) in vacuo. The yield of the third ego penta.fluorophenyl ester of p-nitrophenylsulphenyl-1-glutamine, Nfs-Gln-OPF (4) 7 g (70%) 1 0.05 (2) 0.45 (3). B. difference from amide of o-nitrophenyl-sulfenyl-1-glutamic acid, activated ester Nfs-GlnOPf. like the rest of activated efyram / quickly reacts with an alcoholic solution of ammonia. 1.2 g (8.5 mm) of thiobenzamide was added to a solution of 13.8 g (15.4 mm) of protected pentapeptide (G) in 60 ml of ethanol and, after the reagent was dissolved, 3 ml of acetic acid. The mixture is kept at room temperature for 3 hours and the precipitated yellow precipitate is filtered off. The filtrate is evaporated (40 ° C) in vacuo, the residue is dissolved in a mixture of 20 ml of ether and 85 ml of a 0.1 M solution of sulfuric acid. The ether layer containing benzonitrile is discarded, the aqueous layer is washed with ether to a colorless ether layer () and then at 5 ° C, 2 M sodium carbonate solution is added until the pH of the solution is 9.0. The precipitated amino-pentapeptide oil is separated by Asn-Cys (Trt) -ProLay-Gly-NHa and washed with water (ml). The combined aqueous layer was further extracted (ml) with a mixture consisting of chloroform and ethanol 10 + 1 (by volume), which was added to the oil of the aminopentapeptide. After evaporation of the organic solvents to the solid residue (10.7 g, 13.8 mm, 90 %) aminopeptide peptide was added 25 ml of dimethylformamide, 25 m of dioxane and, after dissolving, 6.4 g (13.8 mm) of HPS-Gln-OPF- (4). The mixture was stirred at room temperature for 0.5 h, then 0.8 ml (7.0 mm) of M-methylmorpholine was added at 15 ° C and stirring at room temperature was continued for 5 hours. The solvents were evaporated. The residue was dissolved in ethyl acetate (100) and the solution was washed successively (with 0.1 M sulfuric acid solution, water, 0.2 M sodium hydrogencarbonate solution, water, dried with sodium sulfate, and ethyl acetate was evaporated (40 ° C) in vacuo. A precipitate formed during extraction The resulting protected hexapeptide (5) is added to the residue after evaporation of ethyl acetate. The residue is triturated with ether (50 ml) and dried in vacuo over phosphorus pentoxide and sodium hydroxide. The yield of the protected hexapeptide (5) is 13.4 g (90% of aminopentapeptide; 85% based on protected Pentapeptide), mp 100-102 cS ° 43.7 (s 1.0; dioxane); Rn 0.03 (4), 0.38 (5). Found: C 57 D4; H 5, 69 ;. N 13.70. SzoloNoOUCH (1025.23). Calculated,%: C 58.58; AND 5, -90; N 13.66. Example 3. O-nitrophenylsulphenyl-1-tyrosyl-1 methyl ester -isoleucine, Nfs-Tyr-Ile-OMe (6). To a solution of 7.4 g (22 mm) of phenylsulphenyl-L-tyrosine o-nitr in 20 ml of dioxane at 15 ° C is added a mixture consisting of 8 ml 3 M solution-dicyclohexylcarbodiimide and 24 ml of 3 M solution of pentafluorophenol in dioxane (an excess of pentafluorophenol is taken to suppress the side reaction of the phenolic hydroxyl group of tyrosine with DCGC). - Stir for 5 minutes, add a solution of 3.5 g (22 mm) of freshly prepared L-isoleucine methyl ester in 15 ml of dioxane and stir the reaction mixture for 2 hours at room temperature. 5.5 ml (50,, mm) of N-methylmorpholine is added dropwise and the mixture is left overnight at room temperature. The precipitated dicyclohexylcarbamide precipitate is filtered off and the filtrate is evaporated in vacuo to an oily residue, which is dissolved in 100 ml of ethyl acetate, and the resulting solution is successively washed (ml) with 0.1 M sulfuric acid solution, water, 0.2 M sodium hydrogencarbonate solution, and dried with sodium sulfate and ethyl acetate is evaporated in vacuo. The dark yellow oil is stirred () with petroleum ether (ml). The solvent is removed by draining, and after the second washing, the precipitate is further dried in vacuum. The yield of solid protected dipeptide (6) 9.5 g (85%); R 0., 45 (2). A small part (5 g) of the dipeptide was dissolved in benzene (2 ml) and the solution was applied on a column (cm) filled with silica gel. Elute with benzene to colorless eluate, then the main fraction is washed out with ether. The ether is evaporated and the yellow residue is triturated with petroleum ether (ml). The yield of the protected dipeptide after chromatography is 82%; mp pl.YU-IO S; .f - 3.4 ° (c 1.0; dioxane). Found,%: C 57.35; H 6.25; N 8.50. . , (461.54). Calculated,%: C 57.25; H 5.90; N 9,10. S-trityl-2-mercaptopropionic acid pentafluorophenyl ester, MprTyr) -Opt (7). . To a suspension of 8 g (23 mm) of S-trityl-2-mercaptopropionic acid (prepared by reacting 2-mercaptopropionic acid with triphenylcarbinol in acetic acid) in 50 ml of dioxane is added a mixture consisting of 8 ml of 3 M solution of DCC. in dioxane and 8 f / tn 3 M solution of pentaft .orphenol in dioxane. The reaction mixture is stirred for 6 hours at room temperature. During this time, the 5-trityl-2-mercaptopropionic acid precipitate gradually dissolved and dicyclohexylcarbamide precipitated, which was filtered, and. washed with dioxane (ml). The filtrate is evaporated in vacuo to an oily residue, 50 ml of petroleum ether are added and the mixture is left at O-C for 24 hours. The precipitated pentafluorophenyl ether (7) is precipitated and dried in vacuum over phosphorus pentoxide and sodium hydroxide. The yield of pentafluorophenyl ether (7) is 10.5 g (90%); m.p. 77-79C; , 85 (1) (yellow spot after spraying the plate with trifluoroacetic acid) .. After recrystallization from methanol, m.p. 82-83p. - Found,%: C 65,74; H 3.66; N 0.12. C, j8Hj9 (514-, 52). Calculated: C 65.36; H 3.72; N 0.10. Methyl 5-trityl-2-mercaptopropyl-1-tyrosyl-1-isoleucine, Mpr (Tr-g) -Tir-Ile-Oms (8). To a solution of 9.2 g (20 mm) of the protected dipeptide (6) in 40 ml of ethanol was added 1.5 g (11 mm) of thiobenzamide and, after dissolving the reagent, 2 ml of acetic acid. The mixture is kept at room temperature.

3 hours and the precipitated yellow precipitate is filtered off by-products. The filter is evaporated in vacuo, the residue is dissolved in a mixture of 30 ml of ether and 105 ml of 0.1 M sulfuric acid solution. The ether layer is separated, the aqueous layer of the aminodipeptide sulfate Thir-Ile-OMe is extracted, ether to a colorless ether layer (5) 3.0 ml), then 2 M sodium carbonate solution is added until the pH of the solution is 9.0. The precipitated aminodipeptide oil is extracted with ethyl acetate (2x20 ml). The ethyl acetate layer was washed with water (ml), dried with sodium sulfate, and the solvent was evaporated in vacuo. The residue, oils (5.9 g, 18 mm, 90%) are dissolved in 30 ml of dioxane and 9.3 g (18 mm) of MnR (Trt) -Off (7) - are added to pactBOpy: After dissolving the activated ether is added dropwise with 1.3 ml (13 mm) of M-methylmorpholine. The mixture is stirred for 6 hours at room temperature, the solvent is evaporated and the residue is dissolved in 100 ml of ethyl acetate. The ethyl acetate solution was washed successively (ml) with a 0.1 M solution of sulfuric acid, water, 0.2 M sodium hydrogencarbonate solution, water, dried with sodium sulfate, and ethyl acetate was evaporated in vacuo. The oily residue is stirred (35 ° C) with petroleum ether (ml). The solvent is removed by draining, and after the second washing it is additionally evaporated in vacuo. Yield of solid protected tripeptide Mpr (Trt) -Tir-леle-OMe (8) 10.8 g, | 90% based on Mpr (Trt) -Opf; ; 85% of the calculation for Nfs-Tir-Ile-OMEZ; mp.108-110 ° C; Rf. 0.42 (2) (orange) after spraying the plate with Pauli reagent. . After re-precipitation from ether with petroleum ether so pl. 110-111 ° C; (D °, 2 (s 1.0 dioxane). Found,%: C71.0b; H 6.61; N 4.26.

 Ce, Nl2 N0.055 (638.83).

Calculated,%: C 71, 45; H 6.63;

  N 4.38. 5-trityl-2-mercaptopropionyl-1. -tyrosyl 1-isoleucin, Mpr (Trt) -Tir-Ile (9),

10.7 g (16.7 mm) of the tripeptide methyl ester (3) are dissolved in 60 ml of dioxane and at room temperature (20 ° C), with stirring, 33.4 ml of a 0.5 M solution of HaijpHH hydroxide is added, then dropwise m for 2 h, another 37 ml of a 0.5 M solution of hydro. sodium oxide. The mixture is stirred for 0.5 hours, then by adding a .1 M solution of sulfuric acid, the medium is brought to a pH of 7.0 and the solvent is evaporated (C5c) to 1 / initial volume of the solution. The residue is brought to 60 MP with water,

A 2 M solution of sodium carbonate is adjusted to pH 8.0 and ethyl acetate (3 x 15 ml) is extracted with residues of tripleptide methyl sulfonate (8). The aqueous layer containing the sodium salt of the tripeptide acid Mpr- (Trt-Thyr-Ile-ONa, is acidified with 1 M solution of sulfuric acid to pH 3.0. The tripeptide (9) is extracted with ethyl acetate (ml). The ethyl acetate layer is washed with water (ml), dried over sodium sulfate and the solvent is evaporated in vacuo. The oily residue is precipitated from ether (20 ml a) adding petroleum ether (60 ml). The precipitate is washed, it is washed on the filter with petroleum ether (2Х.20 ml) and dried under vacuum (0.1 mm Hg). Tripeptide yield (9) 7.4 g (70%) (2); 148-150 ° C, (c 0.5; dioxane); R 0.30 (2) (orange spot after the Pauli reagent was sprayed on the plate). Found: C 70.87; H 6.37;

N 4.48.

Cr, 7 × V (624.81).

C 71.13; H 6.45; Number,%:

N .4,48.

four. . 5-Trityl-2-mer Example of captopropionyl-1-tyrosyl-1-isoleucyl-1-glutaminyl-1-asparaginyl-5-trityl-1-cysteinyl-1-prolyl-L-leucylglycylamide, Mpr (Trt) -Tyr-Ile Gln-Asn-Tsis (Trt) -Pro-Leu-Gli-MN (10).

Claims (3)

0.93 g (6.7 mm) of thiobenzamide and, after dissolving the reagent, 3 ml of acetic acid are added to a solution of 13.3 g (13 of them) of the protected hexapeptide (5) in 60 ml of ethanol. The mixture is kept at. room temperature for 3 hours and filtered precipitated by-products. The filtrate is evaporated in vacuo; the residue is dissolved in a mixture consisting of 20 ml of ether and 75 ml of 0.1 M sulfuric acid solution. The aqueous layer is extracted with ether until the color of the ether layer (ml) is added, then a 2 M carbonate solution is added at 5 ° C. The pH of the aqueous layer is 9.0. The precipitated amino hexapeptide oil Gln-Asn-Cys (Trt) -Pro-Leu-Gly-HH is separated, and washed with water (2.10 ml). The aqueous layers are additionally extracted (3x20 ml) with a mixture consisting of chloroform and ethanol 10 + 1 (by volume), which is added to the aminohexapeptide oil. Organic solvents, solid residue (10.7 g, 11.7 mm, 90%) of amino hexapeptide are quickly evaporated (30 ° C); when dissolved in 30 ml of dimethylformamide, a solution of 7.3 g (11.7 mm) of acid is added the tripeptide (9) in 20 ml of dioxane and another mixture consisting of 4 ml of a 3 M solution of DCC in dioxane and 12 ml of a 3 M solution of pentafluoropheno in dioxane. The mixture was left at 10c overnight. The precipitated dicyclohexylcarbamide precipitate is filtered off and washed on the filter with dioxane (MOT ml). The filtrate is evaporated in vacuo to an oily residue, to which 50 ml of ether are added. The precipitate of the protected nonapeptide (10) is filtered off, washed with ether (3tlO ml) and dried in vacuum over phosphorus pentoxide and sodium hydroxide. The output of the protected nonapeptide (10) is 13.4 g (78% based on tripeptide acid; 70% of calculation on the protected hexapeptide;); 148-150 ° C; R, 0.04 (4) 0.67 (5) (detection by Pauli's reagent and chlorobenzidine reagent). A small portion (0.5 g) of the protected nonapeptide (10) is triturated with hot ethanol (5 ml). After cooling, the precipitate is filtered off (0.4 g) and dissolved in methanol (5 ml). The methanol solution of the protected non-peptide is applied onto a column (3 x 12 cm) filled with silica gel and eluted with methanol (50. ml). The fractions K., which, according to analysis by thin-layer chromatography I, contain the protected nonapeptide, are combined, the solvent is evaporated in vacuum, Residue. they are washed with ether and then with water and dried under vacuum over phosphorus pentoxide and sodium hydroxide. The yield of protected nonapeptide when cleaning 50% so pl. 15 4-15 5 С-, 35, .0,5; dioxane). Found,%: C 65.58; H 6.40; N 9.99. Cai Hj5g N1, (1478.86). . Calculated,%: G 65.79; H 6.47 N 10.42. Cyclic disulfide (1-6) - captopropionyl-and-tyrosyl-1-isoleicill-1-glutaminyl-1 asparaginyl-1cis-teinel-L-prolyl-1-leuidylgly, ilamide desaminooxytocin, Mpr-Ile-Ile-Gln-Asn-bis -Pro-Leu-Gly-MN, 2 (11). 2.21 g (1.5 mm) of the protected nomapeptide (10) are dissolved in 2 liters of an 80% aqueous solution of acetic acid and 0.9 g (7.0 mg-atoms) of iodine is added in one step. in 0.2 l of 80% aqueous acetic acid. The mixture was stirred at room temperature for 1 hour. Then the yellow-brown solution was titrated with a 0.1 M aqueous solution of sulfur dioxide until discolored, and evaporated () c. vacuum to 1/5 of the initial volume. The solution is passed through a column filled with anion exchanger AB 17x8 in OH or acetate form (20 ml). The anions are washed with water (ml) and the combined eluate is evaporated to a small volume (5 ml) and lyophilized. The resulting powder containing desaminooxytocin and triphenyldicarbinol is triturated with ether (ml) and the residue (1.1 g) is purified by partition chromatography on Sephadex in the system n-butanol + benzene + water containing 3.5% acetic acid and 1, 5% pyridine (1 + 1 + 2). The yield of desaminooxytocin (11) is 0.44 g (30% based on the protected nonapeptide), mp. 176-182 ° C; | o (- 90 ° (with 0.5; 1.0 M aqueous solution of acetic acid). Uterotonic activity in rats (in the absence of magnesium ions) is 600t70 international units in mg of the substance. Example 5. Clarification of the time of cleavage of the NFS group in NPS-peptides.... SO MKMOJib of the corresponding peptide (see table) is dissolved in 0.4 ml of ethanol, 8 mg (58 µmol) of thiobenzamide and 0.02 ml of acetic acid are added. The mixture was diluted at room temperature (20 ° C) and at intervals of 10 min, 1 µl of the solution was taken and chromatography was carried out on Sylufol plates. Refining a reference solution containing 5 µmol of peptide in a mixture of O, 4 ml of ethanol and 0.02 ml of acetic acid. The cleavage time of the BFS group (no more than 95% of the initial NSF peptide) is determined by comparing the extracts of yellow spots of those of the original peptide and reference solution. The comparison is carried out using an instrument, RT 65 M (GDR) with a filter 400 named after NFS-GLN-ASN-Cys (Trt) -Pro-Leu-Gly-amide Nfs-Asn-Cys (Trt) -Pro -Ley-Gly-a. Imid NFS-Cys (Trt) -Pro-Leu-GlyChromatographic systems: 1 - benzene, 2 - ether, 3 - ethyl acetate, 4 - chloroform - methanol - acetic acid 85 + 10-f5 (ml) ; -Rf tiob nzamida yellow spot 0.5 (1); 0.65 (2) 0.72 (3). 0.83 (4). .7 Example 6. Cleavage of the NPS group from NFS-amino acids by the action of thiobenzamide or thioacetamide. 28 mg (100 µmol) of Nfs-1-leucine are dissolved in 0.4 ml of ethanol; 8 mg (58 µmol) of thiobenzamide and 0.02 ml of acetic acid are added. Time The removal of the NFS group is determined as previously described; it is less than 10 minutes. I NFs-1-Leucine In system (2) 0.8. The Nfs group in nfs-1-leidine was repeated with 4.4 mg (59 µmol) of thioacetamide. The shredded NFS-gum akJKe is less than 10 min. ; . .. The formula of the invention .. 1. The method of splitting off the sulfenyl groups from N-sulfenyl amino acids and M-sulfenyl peptides by action on the protection by tyrollo S yTTKsii N-sulfa Nylamine acids and N-sulfenyl peptides. thioamdyd and medium 6ganich: esko1 6 solvent in the presence of the liquid, o / tl and h and n and with the fact that, with the Purpose of simplifying the process. , use 0.5-0, 6 mozh of tyoamide per 1 mol of M-sulphate. derivative. ; 2. The method according to p, 1, o, t tea u u and c i; By removing an o-nitrophenylsulfenyl group as the sulfenyl group. 3. The method according to Claim 1, which is based on the fact that thioacetamide or thiobenzamide is used as the tisamide. . . - ...; -1 4. The method according to claim 1, whether or not with the fact that they use N-sulfenyl amino acids and M-sulfenyl peptides that are protected by the thiol function by a 1-trityl group. Information sources taken into account in the examination of l..Synthese von fepttden. (Hg.E.Wunsh) iTeiG 1, Methods der OrganTschen with hem Ie, Houben We f I, (Hg.E.), Bd 15/1, G.Th1 erne, Stutaart, 1974, p.203. 2. Moroder L., Marchiori F., Borin G Scoffon e. Studies on Cytochrome C.Partu, Synthesis of the Protected Heptapeptide (Sequence 17-23) of Bakers Jast so-I-cytochromec, Biopolymers, 1973, 12, c .493 (prototype). 3.Kessler W.JseUn B., SeIektive spaltunq subtituier.ter Phenylsylf eny I-Schu tzg ruppen bei Peptidsynthesen, Helv. Chlm. Acta, 1966, 49, C.1330.