SU730691A1 - Method of preparing dinucleotides - Google Patents

Description

This invention relates to an improved method for the production of dinucleotides that are used to synthesize nucleic acid fragments necessary for research in molecular biology, biochemistry, and genetic engineering. A known method for the preparation of dinucleotides, including cyanoethyl (preparation of a monocyanethyl derivative) nucleotide with an excess (18-60 times) of cyanoethanol and the presence of a condensing agent, dicyclohexylcarbodiimide (DCC); The cyanoethyl derivative was isolated from the reaction mixture by extracting DCC with cyclohexane and precipitating the derivative from the solution with sulfuric ether at -10 ° C, followed by condensing the obtained cyanoethyl derivative of the nucleotide with the nucleotide component in the presence of a condensing agent, triiopropylbenzenesulfonyl chloride (TPN). A disadvantage of the known method is the use of the condensating agent DCC, which has pronounced allergenic properties (its pairs and ether solutions are especially dangerous), and the explosion and fire hazard of the process due to the use of large amounts of flammable solvents (sulfur ether and cshsehexane). to remove DCC). The purpose of the invention is to simplify the process. The aim of the invention is achieved by using a 2-3 fold excess of cyano ethanol in the method for producing dinucleotides, and triisopropylbenzenesulfonyl chloride is avoided as a condensing agent of the cyanoethnlyation stage, which avoids the use of the process. isolating the cyanoethyl derivative as a result of replacing the extraction with cyclohexane (DCC) and precipitating the ether with conventional chromatography, for example on silica gel c. chloroform-methanol system.

In addition, triisopropylbenenesulfonyl chloride or mesitylbonsulfonyl triazolide (MCT) are used as phenyl (lower) alkyl derivatives.

The yield of the target product 32-4O%

Example. 1. Synthesis of 5-phosphoryl deoxythymidilyl- {3 -5) deoxythymidine 61 (pT-T) using dithium ethyl ester (dCE pT) obtained using TPS.

a) Cyanethylation of pT.

A mixture of 1.37 g (4 mmol) of deoxythymidine-5 phosphate (5pT), 0.68 ml of cyanethinol (CEON) is dried by evaporation with absolute | 1M pyridine.

The residue after evaporation is dissolved in 16 ml of pyridine, 3.64 g (12 mmol) of TPS is added to the solution and the reaction mixture is shaken for 2 hours at room temperature under conditions that exclude the ingress of moisture. While cooling to 0 ° C, 16 ml of preferred but cooled water was added to the mixture, and kept for 3 hours at. The reaction mixture is concentrated in vacuo to a minimum volume and evaporated three times with 30 ml of water. 45 ml of water are added and the precipitate is filtered off. The filtrate is evaporated to dryness, dried by evaporation with benzene. The residue after evaporation is dissolved in 40 m chloroform-methanol mixture (9: 1V / v) and applied to the column with 2OO ml of silica gel (o, 140-0.315 mm). Chromatograph first in a linear gradient of methanol in chloroform (4OO ml CHC & 4OOm CHCfc -, 8: 2 V / v), then 400 ml of the mixture (CHSED -, l: lV / v collecting fractions of 8 ml / 10 min. Fractions 70-90 (32,000 O.Egg-) are evaporated to give an oily residue containing 2.7 mmol of SCrT (67.5% in the calculation of tar).

b) Condensation of the nucleoside component (QCEgpT) with the nucleotide component (9pT-OAc).

1.9 g (4.2 mol) of 3-O-acetylthymidine-5-phosphate (YRT-OAc) are dried by evaporation with absolute pyridine. The residue is dissolved in 10 ml of pyridine, 4 g (13.2 mol) TPS is added to the solution and the reaction mixture is maintained.

1-3 hours at room temperature under conditions that prevent ingress of moisture. To the obtained active derivative of the nucleotide component, a solution of 2.7 molceCE RT in 6 ml of Pyrina is added and the mixture is held at room temperature for 6 hours. While cooled, 20 ml of water are added to the reaction mixture and kept at room temperature for 12 hours. Cool the reaction mixture until 48 ml of 2N solution cooled to O ° C are added. N & OH and incubated for 45 min at. Neutralize the reaction mixture ZOO ml

KRS-2P (fN) resin to pH 8, transfer the mass to the column with 100 ml of KRS-2P resin (P ,.) and wash the column with 1.5 l of a 30% pyridine solution in water. The eluate is evaporated to dryness, dissolved

in 5OO ml of a solution of triethylammonium bicarbonate (TEAB) and applied to a column with 2OO ml of ion-exchange resin DEAEmolselect A-25 (HCO 2.6: 34 cm). Chromatograph in a linear gradient

TEAB (3 l 0.05 M - 3 l 0.35 M), collecting fractions of 2O ml / 2O min. 26OOOO.E.g of dinucleotide 9 (pT-T) was isolated from fractions 276-350. Yield 32% (calculated TdT). Р {(рТ-Т) relatively

drT in the EtOH system (3: 7) 0.9; B250 / 250 0.66; ,, 72; E2gQ / E. 26. The content of nucleotide material in the resulting product is 94.3%, the content of dinucleotide according to microcolumn ion exchange chromatography is 97%.

Example 2. Synthesis of 5-phosphoryl-N-anisoyldeoxycytidilyl- (3 -5) -H-isobutyryl-deoxyguanosine 5 (pC G) | using monocyanethyl ether (SSRC) obtained by TPN.

a) Cyanethyl 9pc.

Claims (1)

A mixture of 1.66 g (3.2 mol) of the pyridine salt L - anisoyldeoxycytidine-5-phosphate (JpC), 3.2 mmol of tributylamine (TEA) in pyridine and 0.68 ml (9.2 mmol) of cyanethanol (CEON) are dried by evaporation with absolute shpidin. The residue after evaporation is dissolved in 16 ml of pyridine, 3.64 g (12 mmol) of TPS is added to the solution and the reaction mixture is kept at room temperature for 1.5 hours under conditions that exclude the ingress of moisture. While cooling to (-20) C, 16 ml of water are added to the mixture and incubated for 12 hours at 40 ° C. The reaction mixture is evaporated to dryness, the residue is dried by 3-fold evaporation with dioxane, dissolved in 2 O ml of chloroform-methanol mixture (1: l / v), 5 O ml of skipagel are added to the solution. (O, 14 O-0.315 m carefully mix the mass and dissolve the mixture with vacuum. The resulting dry mass is applied to a column with 200 ml of silica gel (O, 140 O, 315 mm). Chromatograph firstly in linear Graz Kent methanol in chloroform (700 ml C-HCY - 700 ml mixtures CHC-Cz-CH, OH, 8: 2 V / V), then ZOO m mixture (CHCt -, 1: 1, V / V), collecting fractions of 1O ML / 1O min. From fractions 55-76 are 147OO OE, "(2i% of the races ete HaQpC f, of the fractions 78-155-320OO O.E.oa ACerS {46% based) b) condensation. nucleoside component (YSErS) with a nucleotide component (YrSd;). A mixture of O, 7 mmolbCERC and 2.18 (3.9 mol) A O-diisobutyryl deoxyguanosine-5-phosphate (flpQi) by evaporation with absolute pyridine was dissolved in 7 ml of pyridine. Add 1.95 g (7.8.-Mol) MCT to the solution and keep the reaction mixture for 5 days. in the dark under conditions that exclude moisture. When cooled to (-2O) -C. 16 ml of 1 m solution of triethylamine (TEA in pyridine and 8 ml of water) are added to the reaction mixture and cooled to 1-1 12 hours at 4 ° C. The reaction mixture is cooled to (-1 O) ° C, 39 ml of it is added. (-b) of a solution, 2N NaOH and hold for 20 minutes at. The reaction mixture is neutralized with 200 ml of KRS-2P resin (PxjH) to pH 8, and the weight of column is transferred from 100 ml of KRS-2P resin (Py H and Wash the column with 1 L of 20% pyridine solution in water. The filtrate is evaporated to dryness, dissolved in 20 O ml of O, O5 M TEA B and applied to the column with 2OO ml of DEA ion exchange resin E-molselekt A-25 (HCO, 2.6-34 cm). Chromatographic in a linear gradient of TEAB in 10% Mr. alcohol (1.5 l O, O5 M 1, 5 l 0.4 m), collected fractions of 12 ml / 8 minutes From fractions 155-220, 1, ZOOO OE dinucleotide Y () was isolated (Yield. 64% (in CALC)); (Q) in the system c; El OH 0.7; 2, jgQ / E2gQ 0.94; E2gQ / E jQQ l, 13. The content of the nucleotide material is 95%, the content of the dinucleotide according to data from anticoncolo, nighttime ion-exchange chromatography is 97%. PRI me R 3. Synthesis of 5-phosphoryl- - H-isobutyryl deoxyguanylyl- (3 / -5) -H-anisoyl-deoxycytosine 3 (pQ-C) -s using monocyanethyl ether (SEDC) obtained by TPN. a) Cyanethyl dpQ Cyan ethylation of N-isobutyryl deoxyguanosine 5-phosphate (dpQ) is carried out as described in Example 2, except that a mixture of 2 g (4, mol) tributylammonium solidarb, 0.68 ml (9.2 mmol) of CEON is held for 1 h with 3.64 g (12 mol) TPS. After processing the reaction mixture, chromatography on silica gel was carried out and eluted with first 700 ml of a mixture of CNS-CH, OH (8: 2, V / V), then with a linear gradient of methanol in chloroform (500 ml of CHCfcg -, 8: 2, 500 ml of CHCErg, -, 2: 3, Y / V), collected (7 ml / 10 min., Min. From the OO fractions, 11,300 OE, "1 7% in the HaapQ calculation, were isolated from the fractions; 19.О-24О-2700О O.E.ge (40%) olCEpQ. b) Condensation of the nucleoside component (6CEpQ) with the nucleotide component (CrSdd). The condensation is carried out as described in Example 26 except that 2.8 g (4.8 mol) of the pyridine salt of 3-O-acetyl-H-anisoyldeoxy-oxidate i-5-phosphate, 1.6 mmol CEpq and 2.51 g (1O, O mole) MCT. After processing the reaction mixture and removing the protective groups, the chromatographic line was prepared in a linear TEAB gradient in 109a alcohol (3 l 0.05 M - 3 l 0.4 m) to collect fractions of 12 ml / 8 min. Out of 155-220, 25000 O.E.QQ dinucleotide a (). Yield 52% (based on ZCE pQ); R 0.7 v systemic Id OAc-EtOH (3: 7, V / v); E, go / E28o 0.87; E, 3500 0.93. The content of nucleotide material is 95%, the content of dinucleos according to microcolumn ion exchange chromatography is 97%. PRI me R 4. Synthesis of dinucleotides (pT-t) without a prior dicyanethyl derivative obtained by TPS. The preparation of the dinucleotide was carried out as described in Example 1 for hours i ........ t. .-; ..:, r / 7:. / 730 except that a solution of the active derivative of the nucleotide nucleus is added to the reaction mixture obtained by reacting 2.07 g (6.4 mmol) of triethylammonium YpT salt and 1 ml (14.9. Mol) of CEON with g (18 mol) of TPS component obtained by the interaction of 5.85 g (13.2 mol) of the pyridine salt IRTOAc with 11.8 g (39: mol) TPA. After decomposition of the reaction mixture as described in Example 16 with 50 ml of water, cyanoethyl and acetyl protecting groups were removed by treating with 60 ml of concentrated KH-OH for 5 hours at. Evaporate the solution and filter the precipitate. The filtrate is diluted with water to 3 liters and applied to a column with 750 ml of the DEAE ion-exchange resin — molselect A-25 (HSO, 40–5 cm). Chromatograph in a linear gradient of TEAB (O, O M 5 L - 0.5 M 5 L) , collecting fractions of 42 ml / 7 mi, n. From fractions 23O-272, the ICOOH 49OOO O.E.gsy of nucleotide a (pT-T) was obtained. Yield 40% (calculated as zaRT). The product is identical in quality to the product obtained in the example. 1 .8 Formula of the invention 1. A method of producing dinucleotides, including cyanethylation of a nucleotide with an excess of cyanethanol in the presence of a condensing agent, followed by condensation of the resulting cyanoethyl derivative of a nucleotide with a nucleotide component in the presence of a phenyl (lower) alkyl sulphonane derivative, as well, if there are no populations with a nucleotide component in the presence of a phenyl (lower) alkyl sulphonane derivative, they can also use the same. , a 2-3 fold excess of cyanoethanol is used, and triisopropylbenzenesulfonyl chloride is used as the condensing agent of the cyanoethylation step. 2, Method pop.1, distinguishing. And with the fact that as phenyl (lower); alkyl derivatives are used trinzopropylbenzenesulfonyl chloride or mesitylphonyltriazolide, Sources of information taken into account in the examination 1. Badashkeeva AG, Berlin Yu.A. and others. Synthesis of oligo- and polynucleotides, CPS, 1973, No. 3, 394 (prototype) .