The method of obtaining the sodium salt of 2-nitro-4-sulfophenyl ester of biotin

 

(57) Abstract:

The invention relates to the field of Bioorganic chemistry and molecular biology and can be used to obtain oligo(poly)nucleotides and biologically active proteins that carry bootieful group. The proposed method is that the sodium salt of 2-nitro-4-sulfophenyl ester of Biotin is produced by the interaction of sodium salt of 2-nitro-4-sulfophenyl with Biotin in the medium of dimethylformamide in the presence of dicyclohexylcarbodiimide (DCGK), Biotin and DCGK injected into the reaction mixture gradually (portions) to achieve the molar ratio of these reactants in salt, equal 1 : (1,4-1,6) : (1,3-1,5) respectively.

The invention relates to the field of Bioorganic chemistry and molecular biology and can be used to obtain oligo(poly)nucleotides and biologically active proteins that carry bitenova group [1-30]

Known methods for producing esters of Biotin which help synthesize biotinylated derivatives of oligo(poly)nucleotides and proteins [1,4-6] is the closest way conductive to the formation of the sodium salt of 2-nitro-4-sulfophenyl ester of Biotin. Synthesis of lead by who and dicyclohexylcarbodiimide (DCGK) at a molar ratio of initial reagents in DCGK, equal to 1:1:1, by the method of [6] similarly, to obtain the sodium salt of 2-nitro-4-sulfophenyl esters of amino acids, and the selection of the product from the reaction mixture is performed using chromatography on Sephadex LH-20. The yield of the target product 80% of this, However, highlight the product is not convenient because the process chromatography is time-consuming and not always possible due to the inaccessibility of the sorbent.

The task of the invention is to develop a method of obtaining sodium salt of 2-nitro-4-sulfophenyl ester of Biotin, requiring no additional phase chromatographic extraction of product.

The proposed method is that the sodium salt of 2-nitro-4-sulfophenyl ester of Biotin is produced by the interaction of sodium salt of 2-nitro-4-sulfophenyl with Biotin in DMF medium in the presence of DCGK, with Biotin and DCGK injected into the reaction mixture gradually (portions) to achieve the molar ratio of these reactants to the sodium salt of 2-nitro-4-sulfophenyl equal 1:81,4-1,69):(1,3)-1,5) respectively.

When carrying out the process in the proposed conditions of the sodium salt of 2-nitro-4-sulfophenyl fully expended on the formation of ester of Biotin. Because the treatment of dicyclohexylcarbodiimide precipitates. Thus, the only compound in the solution is the desired product, recorded in UV-light in the analysis of aliquots of the reaction mixture by thin layer chromatography. To generate a crystal clear enough filtered solution of the reaction mixture from the precipitate, the filtrate was evaporated to, and the residue dissolved in methanol and the product precipitated with ether. The product yield is increased to 92% in comparison with the prototype. When carrying out the process under conditions when the initial reactants in the above ratio immediately introduced into the reaction mixture, i.e., not portions, education is difficult to separate by-product.

Reducing the target number of Biotin (molar ratio of sodium salt of 2-nitro-4-sulfophenyl: Biotin 1:(1,2-1,4)) does not lead to a complete expenditure of sodium salt of 2-nitro-4-sulfophenyl, and increasing the number of dicyclohexylcarbodiimide (final molar ratio of sodium salt of 2-nitro-4-sulfophenyl: dicyclohexylcarbodiimide 1:(1,6-1,9)) leads to the reaction mixture a by-product, which is recorded in UV-light using the method of thin-layer chromatography.

Example 1 (the prototype). Synthesis is carried out according to the method C dissolved in 62 ml of dimethylformamide, and then add 44,6 MK mol of Biotin. It chilled in ice, the reaction mixture is added to 43.2 MK mol of dicyclohexylcarbodiimide and incubated for 1 hour at 0oC and 21 hours at room temperature under stirring. An aliquot of the reaction mixture is applied on the thin-layer plate of silica gel and spend chromatography in the system ethanol: chloroform (6:4). The products formed in the reaction is recorded in the form of spots on the plate in UV light. The analysis shows that when carrying out the reaction under such conditions (molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide is 1:1, 08:1,04 respectively) in the reaction mixture appears the product with Rf0,35, and sodium salt of 2-nitro-4-sulfophenyl not hitting.

Example 2. Carry out the same procedure with the same quantities as in example 1, and then the reaction mixture was again cooled in ice, add 20,8 MK mol of Biotin, an 18.4 MK mol of dicyclohexylcarbodiimide and again allowed to stand for 1 hour at 0oC, 21 h at room temperature and 12-15 hours at +5oC. (Final molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide is 1:1, 58:1,49, respectively). Analysis the Alize detects a complete transformation of the sodium salt of 2-nitro-4-sulfophenyl in the product with Rf0,35. Sediment dicyclohexylamine and neproreagirovavshikh Biotin is separated by filtration, the filtrate is evaporated to an oily residue, the residue is dissolved in 50 µl of methanol. The product precipitated their methanol ethyl ether and dried over P2O5in a vacuum. The output 80%

Example 3. Perform all the same stages as in examples 1 and 2, except that the reaction mixture in the amount of 420 μl initially contains 264 MK mol of sodium salt of 2-nitro-4-sulfophenyl, 278 MK mol of Biotin and 269 MK mol of dicyclohexylcarbodiimide, and then it was added 96 MK mol of Biotin and 102 MK mol of dicyclohexylcarbodiimide. (Final molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide is 1:1, 4:1,39, respectively).

The resulting product was analyzed by thin layer chromatography in three solvent system: ethanol-chloroform (6:4), B ethanol-water (9: 1 n-butanol:acetic acid:water (4:1:1). The analysis shows that in all systems of the solvents the product is represented by a single spot with Rf0,35, to 0.69 and 0.55 in systems a, B and C respectively. The structure of the obtained product was proved by using the method 1H-NMR spectroscopy. The assignment of the signals was conducted by the doctor) to 1.59(m, 2H, H-7, H-7'); 1,68 (m, 1H,H-6); to 1.82 (m, 3H, H-6', H-8H-8'); and 2.83 (m, 3H,H-3,H-9,H-9'); 3.04 from (DD, 1H,H-2); to 3.41 (m, 1H, H-4); 4,48 (DD, 1H,H-5); the 4.65 (m, 1H,H-1); EUR 7.57 (d, 1H,H-12); 8,21 (DD, 1H, H-11); to 8.57 (d, 1H,H-10). Exit 92%

Example 4. The synthesis of the product is carried out similarly as described in examples 1 and 2, except that the final molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide early 1:1, 42: 1,32 respectively. Analysis of aliquots of the reaction mixture is carried out as described in example 1 finds the full divergence of the sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl ester of Biotin.

Example 5. The synthesis conditions similar to the conditions described in examples 1 and 2, except that the final molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide is 1:1, 41: 1,47 respectively. Analysis of aliquots of the reaction mixture is carried out as described in example 1, shows the full expenditure of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl ester of Biotin.

Example 6. The synthesis conditions similar to the conditions described in examples 1 and 2, except that the final molar sootnoshenie the Liz aliquots of the reaction mixture, are as described in example 1 finds the full expenditure of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl ester of Biotin.

Example 7. The synthesis conditions similar to the conditions described in examples 1 and 2, except that the final molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide is 1:1,52: 1,44 respectively. Analysis of aliquots of the reaction mixture is carried out as described in example 1 finds the full expenditure of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl ester of Biotin.

Example 8. The synthesis conditions similar to the conditions described in examples 1 and 2, except that the final molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide is 1:1,49: 1,51, respectively. Analysis of aliquots of the reaction mixture is carried out as described in example 1 finds the full expenditure of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl ester of Biotin.

Example 9. The synthesis conditions similar to the conditions described in examples 1 and 2, for acylcarnitine is 1:1,58:1,31 respectively. Analysis of aliquots of the reaction mixture is carried out as described in example 1 finds the full expenditure of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl ester of Biotin.

Example 10. The synthesis conditions similar to the conditions described in examples 1 and 2, except that the final molar ratio of sodium salt of 2-nitro-4-sulfophenyl, Biotin and dicyclohexylcarbodiimide is 1:1,57: 1.37. Analysis of aliquots of the reaction mixture is carried out as described in example 1 finds the full expenditure of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl on the formation of sodium salt of 2-nitro-4-sulfophenyl ester of Biotin.

Research in this application was made with support from the International Science Foundation and Russian Government (grants RBN000 and RBN300).

Sources of information:

1. Bayer, E. A. and Wilchek, M. Methods of biochemical analysis, 1980, v.26, p. 1-45.

2. Pantano P. W. G. Kuhr Anal.Chem. 1993, v.65, N5, p.623-630.

3. Badashkeeva A., Zarytova C. F. Molecules. biology, 1989, T. 23, vol.5, S. 1221-1226.

4. Bayer, E. A. and Wilchek, M. Methods in enzymolrgy, 1974, v.XXXIV, p.265-267.

The method of obtaining the sodium salt of 2-nitro-4-sulfophenyl ester of Biotin, characterized in that the sodium salt of 2-nitro-4-sulfophenyl enter in interaction with Biotin in the medium of dimethylformamide in the presence of dicyclohexylcarbodiimide (DCGK), and Biotin and DCGK injected into the reaction mixture in portions to achieve the molar ratio of these reactants to the sodium salt of 2-nitro-4-sulfophenyl 1 1,4 1,6 1,3 1,5 respectively.

 

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