SU721452A1 - Method of preparing oligonucleotide 5'-mono- or polyphosphate amides at terminal phosphate group - Google Patents

Description

The invention relates to an improved method for producing amides of Si mono- or polyphosphates of oligonucleotides at the terminal phosphate group, which are widely used in molecular biology for the study of matrix reactions, especially for complementary targeted modifications of nucleic acids, as well as affinity reagents.

₍ Known methods for producing amides of 5-monophosphates of oligonucleotides at the terminal phosphate group using dicyclohexylcarbodiimide [1] or diphenylchlorophosphate.

Both methods are accompanied by a significant adverse reaction in internucleotide phosphate groups, increasing with increasing length of the oligonucleotide.

There is also a method of producing amides of 5-mono- or polyphosphates of oligonucleotides [h], based on the activation of the terminal phosphate group of 5-mono-phosphates of oligonucleotides by conversion

...... '' it into mixed anhydride by the action of an excess of 2,4,6-hlorangidrvda trimetilbenzoynoy acid in dry pyridine for 20-30 minutes and subsequently reacting the mixed anhydride with amines for 2-W ^x days in water or 18-20 hours in dry dimethylformamide. The yields of both mixed anhydrides and 5-amides of oligonucleotides are 60-90%. The synthesized amides are isolated by paper chromatography. But the synthesis of 51-amvd oligonucleotides is a two-stage process in different solvents, the reaction time of mixed oligonucleotide anhydrides with amines in water is 2-3 days, in dimethylformamide 18-20 hours, i.e. significant, moreover, the synthesis is complicated by a side reaction of internucleotide phosphate groups, leading to intramolecular phosphorylation in the ribo-series

2-hydroxyl, and then to isomerization or rupture of the internucleotide bond.

The aim of the invention is to simplify the process.

This goal is achieved in that the amides of 5-mono- or polyphosphates of oligonucleotides at the terminal phosphate group are obtained by reaction in a mixture of tri- or tetraalkylammonium salts of 5-mono- or polyphosphates of oligonucleotides in a dry polar aprotic solvent (dimethylformamide, dimethyl sulfoxide, pyridine), 3 A 5-fold excess of a triphenylphosphine-2,2'-dipyridyl disulfide condensing agent; and a 5-10-fold excess of an amine (pKa> 5). The process usually takes 2-4 hours at room temperature. Target products are distinguished by conventional methods; precipitation of the reaction mixture in ether or ion exchange chromatography. The yield of the target product in both cases is not less than 80%.

Example 1. Synthesis of 5-piperidide trideoxythymidylate.

210 OE _GB7 (7.1 μmol) of triethyl * ammonium salt (dpT is dissolved in 0.2 ml of dry pyridine, 4 μl (50 mmol) of piperidine, 8 mg (30 mmol) of triphenylphosphine are added to the solution,

6.6 Mg (30 mmol) of 2.2 * dipyridyldi sulfide and incubated for 3 hours at room temperature. The reaction mixture was added to 10 ml of ether, the precipitate was separated by centrifugation, washed with ether (3x5 ml), dried in vacuum. Yield 170 OE _2b7 (88%). According to microcolumn chromatography on DEAE cellulose in the TomlinsonTener system, the preparation contains 96% of the target product and 4% (dpT) ₃ ; according to thin-layer chromatography on Eastman-kodak cellulose plates in the isopropanol: ammonia: water - 7: 1: 2 system, the preparation contains two substances with Rf 0.75 (target product) and Rf 0.41 ( ^d pT ₃ , traces).

Example 2. Synthesis of p-morpholide 5-pyrophosphate trideoxythymidylate.

100 OE (3 mmol) of triethylammonium salt of 5-trideoxythymidylate pyrophosphate (dppTpTppT) is dissolved in 0.1 ml of dimethyl sulfoxide, 2 μl (25 mmol) of morpholine, 5.4 mg (20 mmol) of triphenylphosphine, 4.4 mg are added to the solution (20 mmol) of 2,2'-dipyridyl sulfide and incubated for 2 hours at room temperature. The reaction mixture was diluted with water to 3 ml and

721452 A was extracted with ether (6x2 ml). An aliquot of the aqueous layer is analyzed by paper chromatography in the ethanol-1M ammonium aietate system, pH 7.5 (7; 3). Sbnaru — ₅ wife with two spots with RI 0.24 (target product) and R1 0.08 (dppTpTpT, traces). According to microcolumn chromatography on DEAE-cellulose in the Tomlinson-Tenera system, the preparation (aqueous Yu. Layer) contains 95% of the target product.

Output 82 OE _t6? (82%).

Example 3 Synthesis of 5 ^ -benzylamide dideoxythymidylate.

100 mg (0.11 mmol) of triethylammonium, niya salt (dpT) _{4 is} dissolved in 1 ml of dry pyridine, 100 μl (0.9 mmol) of benzylamine are added to the solution, 66 mg (0.3 mmol) of 2,2-dipyridyldi - ²⁰ sulfide, 78 mg (0.3 mmol) of triphenylphosphine and incubated for 2 hours at room temperature. In the NMR spectrum of the reaction mixture, signals with chemical shifts of + 1.9 ppm are observed.

²⁵ (internucleotide phosphate) and -7.1 ppm (5'-terminal amide ester phosphate), intensity ratio 1: 1, as well as triphenylphosphine oxide signal (-25.7 ppm). The reaction mixture was added to 25 ml of ³⁰ ether, the precipitate was separated by centrifugation, washed with ether (4x10 ml), dried in a vacuum. Yield 100 mg (100%). The homogeneity of the drug is confirmed by GC on a SifufoP plate in the system CHjCN - H ^ O (4: 1), 0.33.

Claims (3)

The invention relates to an improved method for producing amide sLMpHO- or polyphosphates of oligonucleotides at the terminal phosphate group, which are widely used in molecular biology for the study of matrix reactions, especially complementary to the addressed modification of nucleic acids, as well as affinity reagents. There are known methods for producing amides of B-monophosphates of oligonucleotides to the terminal phosphate group using dicyclohexylcarbodiimide 1 or diphenylchlorophosphate 2 Both methods are accompanied by a significant adverse reaction to the internucleotide phosphate groups, which increase with increasing length of the oligonucleotide. There is also known a method of producing amides of 5-mono- or polyphosphates of oligonucleotides Uz, based on the activation of the terminal phosphate group of 5-mono-phosphates OL1X) nucleotides, furthermore, into a mixed anhydride under the action of an excess of 2,4,6-trimethylbenoic acid in dry pyridine for 20-ZO min and the subsequent interaction of the mixed anhydride with amines for 2-3 days in water or 18-2 O h in dry dimethylformamide. The yields of both mixed English and 5-amide oligonucleotides are 6O-9O%. The synthesized amides are isolated by paper chromatography. But the synthesis of 5-amvd oligonucleotides is a two-step process in different solvents, the duration of the reaction of mixed anhydrides of oligonucleotides with amines in water for 2-3 days, in dimethylformamide for 18-20 hours, i.e. This is significant, besides, the synthesis is complicated by an adverse reaction on the internucleotide phosphate groups, leading in the ribo series to intracellular phosphorylation of 2-gvdroksil, and to the isomerization or cleavage of the inter-nucleotide bond. The aim of the invention is to simplify the process. The goal is achieved by the fact that amides of 5-myo- or polyphosphates of rligonucleotides on the terminal phosphate group are obtained by carrying out the reaction in a mixture of 51 mono- or tetra-alkylammonium salt of mono- or polyphosphates of oligonucleotides in a dry polar alroton solvent (dimethylformamide, dimethylsulfoxide, pyridine), 5-fold excess condensing agent - triphenylphosphine-2,2-dipyridyl disulfide, and 5-1O-fold excess amine (). The process usually takes 2-4 hours at room temperature. Target products are isolated by conventional methods; by precipitating the reaction mixture into ether or byObmixing chromatography. The yield of the target product In both cases, at least 80%. Example 1. Synthesis of b piperidide tridesoxythymidylate. 21 O Ojg (7.1 µmol) of triethyl ammonium salt (dpT), dissolved in 0.2 ml of dry shfidina, was added to a solution of 4 µl (5 O mmol) of piperidine, 8 mg (30 mmol) of triphenylphosphine, 6.6 Mg (30 mmol) 2,2-dipyridyl disulfide and incubate for 3 hours at room temperature. The reaction mixture was added to 10 ml of ether, the precipitate was separated by centrifugation, washed with ether (3 x 5 ml), dried in vacuum. Yield 170 OEj67 (88%). According to microcolumn chromatography on DEAE-cellulose in the Tomlinson-Generator system, the preparation contains 96% of the target product and 4% (SpT) jj according to thin-layer chromatography on the itastrnan-koflaK cellulose plates in the isopropanol: ammonia: water - 7: 1: 2 system contains two substances with Rf 0.75 (target product) and Rf 0.41 (dpTj, traces). Example 2. Synthesis of | 5-morpholide 5-pyrophosphate tridesoxythymidylate. 1OO OE (3 mmol) triethylamine; the nium salt of 5 β-trideoxide thymidylate pyrophosphate (dppTpTpT) is dissolved in O, 1 ml of dimethyl sulphoxide, 2 μl (25 mmol) of morpholine, 5.4 mg (2O mmol) triphenylphosphine are added to the solution, 4.4 mg (20 mmol) of 2,2-dipyridylsulfide and incubated for 2 hours at room temperature. Reaction: the mixture is diluted with water to 3 ml and kstragiruyut ether (6x2 ml). The aqueous layer aliquot is analyzed by paper chromatography in an ethanol-1M ammonium adatate system, pH 7.5 (7; 3). Two spots with Rf 0.24 (business product) and 08 (dppTpTpTT, traces) are found. . According to microcolumn chromatography on DEAE-cellulose in the Tomlinson-Tenera system, the preparation (aqueous layer) contains 95% of the target product. Yield 82 OU, 67 (82%). Example 3. Stimulation of 5-benznpamide dideoxythymidylate, 1OO mg (0.11 mmol) of triethylammonium salt (dpT) was dissolved in ml of dry pyridine, added to a solution of 1OO µL (0.9 mmol) of benzylamine, 66 mg (0.3 mmol) 2,2-dipyridyl disulfide, 78 mg (0.3 mmol) of triphenylphosphine and hold for 2 h at room temperature, In the spectrum of the reaction mixture, signals with chemical shift + 1.9 ppm are observed (internucleotide phosphate) and -7.1 ppm (5-terminal amido ester phosphate), an intensity ratio of 1: 1, as well as a triphenylphosphine oxide signal (-25.7 ppm). The reaction mixture is added to 25 ml of ether, the precipitate is separated by centrifugation, washed with ether (4x10 ml), dried in vacuo. The output 1OO mg (1OO%). The homogeneity of the drug is confirmed by 1CA on the SlCufoP plate in the CHjCN-H2.O system (4: 1), R 0.33. Claim 1. Method for producing amides of 5-mono or polyphosphates of oligonucleotides on the terminal phosphate group in a dry polar aprotic solvent in the presence of an amine (), characterized in that, in order to simplify the process, the reaction is carried out in a mixture of tri- or tetra-aplylammonium salt 5 mono- or polyphosphates of oligonucleotides, a 3-5-fold excess of triphenylphosphip-2, 2-dipyridyl disulfide 14 | ida and a 5-1O-fold excess of amine. Sources of information taken into account during the examination 1. Sokolova A., I., Gatinska L. G., Nedby V. V., Smirnov V. D., Ruhl da P. I., Shcheb pova. 3. A. and Prokofiev M. A. 57214526 Synthesis of amides of oligonucs.heotvdov DAN () fesh alalanine pier 4yusphate meSSSR (1971) 196 NO 3 p. 613-615. Method, JOH (1964) 34, p. 359-261. 2. Vorobev O. V., Shabarova Z.A., 3.NucCetc Adds RearchV 1976, Prokofiev M.A. Synthesis of nucleotide 3, fo 4, p. 903-916 (prototype).