Method of removing phenylhydrazides protective group, a carboxyl blocking function

 

(57) Abstract:

Use: in organic chemistry, synthesis of peptides. The inventive method of removing phenylhydrazides protective group, a carboxyl blocking function, by catalytic oxidation phenylhydrazides group oxygen under the action of the complex of copper (II) with low-molecular-weight nitrogen-containing organic ligands at a molar ratio of from 1:1 to 1:500 at a pH of from 4.0 to 6.5. 5 Il., table 1.

The invention relates to the field of organic chemistry, in particular to the field of peptide synthesis, and can be used to remove temporary protective group, a carboxyl blocking function.

Repeatedly attempts to use phenylhydrazine group for temporary protection of the carboxyl functions in the synthesis of peptides. Have been developed mild and selective introduction of this protection with the aid of enzymes. It was also shown that phenylhydrazine group is of type "potentially triggered" protective groups and can be used both for protection and for the subsequent activation of the carboxyl function.

However phenylhydrazine protection has not found wide application is the super hard conditions.

Known methods of removal phenylhydrazides protective group, a carboxyl blocking function. Most of the currently proposed methods of release include chemical oxidation phenylhydrazide to highly unstable phenyldiamine with subsequent spontaneous decay:

-CO-NH-NH-C6H-CO-N= N-C6H5-> -CO-OH + N2+ C6H6As oxidizing agents in the first stage using potassium permanganate, ferric chloride, iodine, bromosuccinimide, manganese dioxide, leads to compounds, which lead, copper acetate. The reaction with ferric chloride proceeds in an acidic environment, which imposes restrictions on the use of tert-Budilnik and tert-butyloxycarbonyl protective groups. The reaction with manganese dioxide leads to the oxidation of methionine by 67% of the reaction with copper acetate occurs at a temperature 96-97aboutC. the Use of iodine can lead to a modification of the cyclic structures of histidine and tyrosine, as well as to the elimination of acetamidomethyl and trailvoy protection cysteine. Bromosuccinimide modifies tryptophan. Tryptophan can also be nonspecific oxidation.

Thus, we must conclude that at present there is no I in peptide synthesis.

Closest to the claimed is enzymatic removal method phenylhydrazides protection using laccase [1] Catalyzed by enzyme release proceeds under mild conditions and without adverse reactions. As the oxidant used the oxygen in the air. The disadvantages of this method are the high cost of laccase [2] and the impossibility of its use for the release of hydrophobic peptides in organic solvents (see Fig. 1).

The aim of the invention is the reduction and expansion of technological capabilities of the method.

The described method of removing phenylhydrazides protective group, a carboxyl blocking function is in the catalytic oxidation phenylhydrazides group oxygen under the action of an organic complex of copper (II) with low-molecular-weight nitrogen-containing organic ligands, resulting from the interaction of copper salts and low molecular weight nitrogen-containing organic ligands, taken in a molar ratio of from 1:1 to 1:500 at pH values of 4.0 to 6.5.

Common features of the described method and the nearest technical solutions are: release, which includes oxidation foilage disintegration;

the release process is catalytic, and the catalyst is an organic complex of copper (copper is part of the active centre of laccase);

the oxidizing agent is oxygen.

The advantages of this method are:

deblokiruyuschee reagents (salts of bivalent copper and organic nitrogen-containing ligands, such as pyridine), which are cheap and available anywhere, including preparative, quantities;

the release process now proceeds in an aqueous-organic medium with a high content of dimethylformamide, which allows reaction with hydrophobic peptides in concentrated solutions that simplify the extraction (see Fig. 1).

The method is as follows. The peptide that contains the deleted phenylhydrazine group, dissolved in dimethylformamide, add an aqueous solution of salts of bivalent copper, an aqueous solution of organic nitrogen-containing ligand, and the reaction mixture was stirred at room temperature under conditions ensuring free access of air. The course of the reaction is controlled, for example, by thin layer chromatography. Upon completion of the reaction is released Ph under the action of lactase from dimethylformamide concentration in the reaction mixture; in Fig. 2 the dependence of the concentration of the original model Tripeptide Phe-Trp-Gly NHNH Ph with time during the test release catalyzed by complexes of copper (II) with an organic nitrogen-containing ligands: a pyridine, b-imidazole, N-Mei, g without ligands, Fig. 3 the degree of release of the model Tripeptide Phe-Trp-Gly NH NH Ph under the action of pyridinium complex of copper (II) from dimethylformamide concentration in the reaction mixture, and Fig. 4 spectrum of proton magnetic resonance fully protected Tripeptide Z-Gly-hys(Boc) - Leu NH NH Ph (spectrum a) and partially released Tripeptide Z-Gly hys(Boc)-Leu NH NH Ph (range B) of Fig. 5 same as in Fig. 2, using as the catalyst complex of copper (II) with lysine.

P R I m e R 1 (comparative). Enzymatic release model Tripeptide Phe Trp Gly NH NH Ph is carried out in a mixture of water, dimethyl formamide with different content of dimethylformamide. The reaction mixture containing 1 mm of peptide 2 on. dimethyl sulfoxide, 20 μl of a solution of laccase with specific activity of 800 units/ml, nitroacetate buffer, 0.2 M, pH 4.0 and dimethylformamide. Total volume of reaction mixture 1 ml of the reaction control method ivash. In Fig. 1 presents the dependence of the article the catalytic activity of laccase significantly suppressed in the presence of dimethylformamide. Therefore, the method enzymatic removal phenylhydrazides protective group limited is applicable in the case of hydrophobic peptides.

P R I m m e R 2. Release model Tripeptide carry oxygen in the presence of copper complexes with nitrogen-containing organic ligands. The reaction mixture (total volume 1 ml) containing 1 mm peptide, 0.4 mm of chloride of copper (II), 0.5 ml of dimethylformamide, 0.5 ml of 0.2 M nitroacetate buffer, pH 4.0 and 20 mm of the organic ligand. The reaction mixture was stirred under conditions ensuring free access of air. The reaction control method ivash. In Fig. 2 presents the kinetic curves showing the process of release of the peptide in time. It is seen that the process release oxygen catalyzed by copper ions, significantly accelerated in the presence of nitrogen-containing organic ligands.

P R I m e R 3. The method is carried out analogously to example 1, but as the catalyst use is not a laccase, and CuCl2copper (II) and pyridine in concentrations of 0.4 mm and 20 mm respectively. In Fig. 3 shows the dependence of the degree of release of the model Tripeptide Phe Trp Gly NHNH Ph for 30 minutes on the concentration of dimethylformamide in reallycool concentration of dimethylformamide (cf. with the known method, Fig. 1).

P R I m e R 4 (preparative). 6.8 g fully protected Tripeptide Z-Gly-Lys (Boc)- Leu NHNHPh (fragment 10-12 calcitonin salmon) dissolved in a mixture containing 425 ml of dioxane, 212 ml of 1 M peridiniaceae buffer 106 ml of glacial acetic acid and 10 ml of 0.2 M solution of CuCl2. The mixture is intensively stirred for 20 h at room temperature in conditions that ensure free access of air. Upon termination of the reaction mixture is evaporated to dryness on roton evaporator and dissolved in a mixture of 400 ml of ethyl acetate and 300 ml of 1 N. sulfuric acid. The aqueous layer was discarded, the organic layer washed with 200 ml of 1 N. sulfuric acid and water until neutral. The organic layer is evaporated and the oil obtained is crystallized from a mixture of diethyl ether and hexane. The output is partially released Tripeptide Z-Gly-Lys(Boc) - OH Ley 5,63 g (92.3 per cent).

P R I m e R 5. The method is carried out analogously to example 2, but instead of dimethylformamide use other organic solvents or process carried out in aqueous medium in the absence of organic solvents. The results of the analysis of the reaction mixture after 30 min after start of the reaction are shown in table.

The table shows that the method is implemented in water-bodies is Ino, the nature of the organic solvent does not apply to signs that are essential for the implementation of the method.

P R I m e R 6. The method is carried out analogously to example 2, but as a ligand using lysine. For 30 min according to ivash conversion rate is 5% From the above example shows that in the presence of lysine as a ligand, the method is implemented, although the catalytic activity of the complex of copper (II) with lysine is lower than in the case of the complex of copper (II) with pyridine.

The ratio of copper (II) ligand can vary within wide limits, as evidenced by the results shown in example 7.

P R I m e R 7. The method is carried out analogously to example 2, except that as a ligand using pyridine or imidazole at a concentration of from 0.4 mm to 0.2 M. the Results are presented in Fig. 5. From the graph we see that the ratio of copper (II) ligand can be varied in the range from 1:1 to 1:500.

METHOD of REMOVING PHENYLHYDRAZIDES PROTECTIVE GROUP, a CARBOXYL BLOCKING FUNCTION, including catalytic oxidation phenylhydrazides group oxygen in the environment of a solvent in the presence of initiator, characterized in that the initiator uses the complex of copper (II) neither usrnam nitrogen-containing organic ligand in a molar ratio of 1 to 1 500 at pH values of 4.0 to 6.5.

 

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