SU1759842A1 - Method of producing 5ъ-0-dimethoxytrithylthymidene-3ъ-hydrophosphonate - Google Patents

Abstract

Use: as a monomer for oligonucleotide synthesis. The essence of the invention: the product - 5-0-dimethoxytritylthymidine-31 - hydrophosphate. BF S15N25№OvR. OUTPUT 91%. Arm ke 269 NM

Description

This invention relates to the field of bioorganic chemistry, in particular, to an improved process for the preparation of 5 -0-dimethoxytrityl-mitidine-3-hydrogenphosphonate (Dmtr Three).

This compound is used as a monomer for the chemical synthesis of oligonucleotides widely used in the field of molecular biology and genetic engineering.

A known method for producing Dmtr Trn 1, consisting of 3 stages. In the first of these, 5 -0-dimethoxytritylthymidine (Dmtr T) is reacted with salicyl chlorophosphite (Sal RS) with mixed indian-pyridine at room temperature for 10 minutes, in the second stage, the reaction mixture is treated with water for 5 minutes and the product is isolated using known methods with a yield of 88%. The disadvantages of this method are the high laboriousness and unnecessarily long auxiliary stages of the process due to the need to obtain an intermediate product, Sal PCI, commercially unavailable and labile during storage, as well as subsequent difficulties in cleaning the product from salicylic acid before the reaction.

The closest to the invention is a method consisting of 4 stages 2. In the first stage, an intermediate product, triimidazolylphosphine, is obtained by reaction of phosphorus trichloride (), imidazole and triethylamine in acetonitrile at 0 ° C for 15 minutes, in the second stage the product obtained process the Dmtr T solution in acetonitrile at the same temperature for 30 minutes, then the mixture is hydrolyzed for 54 minutes and the product is isolated by known methods with a yield of 86%.

The disadvantages of this method are the increased laboriousness, the excessive duration and complexity of the process due to the step of obtaining the intermediate tririmidazolylphosphine, as well as the time spent on hydrolysis of the reaction mixture and purification of the target product from large quantities of imidazole, which is formed. In addition, high

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Ke cost of imidzola (500 rubles / kg) increases the cost of the target product.

The aim of the invention is to simplify and speed up the process, as well as increase the yield of the target product.

The goal is achieved by the fact that a suspension of 5 -0-dimethoxytrityl thymidine (Dmtr T) in acetonitrile is treated with PCl3 in the presence of a low-basic tertiary amine (pKa less than 8) with stirring. The tertiary amine is preferably selected from the group: pyridine, quinoline, 2,6-lutidine, 2,4,6-collidine, N. N-dimethylaniline.

The essence of the method is as follows.

A suspension of Dmtr T in acetonitrile is treated with stirring and low temperature, preferably at 0 - (- 20) ° C in the presence of a low basic tertiary amine (pKa less than 8) selected from the group: pyridine, quinoline, 2,6-lutidine, 2.4 , 6- collidine. Kj.-dimethylaniline, etc., for 3-10 min according to the scheme DmirO 0ThyB Dwlr04 QTh9

U + PC1 SheyG V

it, ci

(DmlfT) RF

Ul

(DmtrTpc,

where Dmtr is dimethoxytrityl; Thy, thymine-1-yl; B - tertiary amine.

Then the reaction product is hydrolyzed by adding aqueous pyridine at the same temperature for 1-2 minutes according to the scheme

Dmt.rO DmlrTpc, 2iWX V ° J9

S-GH -

fDmlrT °

where Py is pyridine, and the product is isolated by known methods. The yield of the target product is 90-92%. The time spent at the main stages of the process is 4-12 minutes, depending on the temperature and type of base.

The salient feature of the proposed method is the treatment of a suspension of b -O-dimethoxytrityl thymidine in acetonitrile with phosphorus trichloride in the presence of a low basic tertiary amine.

The use of a low-base tertiary amine in p «a less than 8 instead of a mixture of secondary and highly basic tertiary (pKa 10) amines in the prototype allows, firstly, to avoid the stage of conversion of the active to less reactive derivatives of the type of tririmidazolylphosphine, which simplifies and accelerates the process and, secondly, see action,

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preventing its attack of the heterocycle in the Dmtr T molecule by reducing the ionization of the functional groups of this part of the molecule in the presence of a low basic amine.

The use of a suspension of Dmtr T in acetonitrile (instead of its solution in the same solvent) allows you to maintain a constant excess over the course of the reaction with respect to Dmtr T, which prevents the formation of dimeric side products like (Dmtr TfcPCI and provides a high yield of the target product.

The invention is illustrated by the following examples.

Example 1. Synthesis of β-O-dimethoxytritylthymidine-3 -hydrophosphonate in the presence of pyridine. A suspension of Dmtr T (0.544 g, 1 mmol) in acetonitrile (4 ml) and pyridine (0.24 ml, 3 mmol) is treated (0.16 ml, 1.8 mmol) at -15 ° C and stirred for 10 minutes until dissolved. the precipitate, the solution is hydrolyzed with water-pyridine (1: 3 by volume, 4 ml) at the same temperature for 1 min with stirring, a 1 M solution of triethylammonium bicarbonate (pH 7.5) (7 ml) is added, shaken and extracted with chloroform (3x15 ml). The combined extracts are washed with water (5 ml), dried with sodium sulfate and, after filtering off the drying agent, the filtrate is evaporated in vacuo, adding 0.5 ml of triethylamine to it. A solution of the product in chloroform (3 ml) and pyridine (0.1 ml) was applied to a silica gel column (20 g) (CHFR, 40-100 µm) and the product was eluted with a stepwise gradient of ethanol in chloroform containing 1% pyridine: 10% , 15%, 20%. 25% (50 ml each). The product is collected (10500 o.e.267), the solvents are evaporated in vacuo. The residue was dissolved in chloroform (4 ml) and ether (2 ml) and poured into ether-pentane (1: 2 by volume, 80 ml). After centrifuging and drying in vacuo over P205, 0.64 g (91%) of Dmtr Trn is obtained in the form of triethylammonium salt. The product is homogeneous at TLC on silica gel in the systems: chloroform-ethanol (4: 1), ethanol-chloroform-1 M ammonium acetate, pH 6 (10: 10: 1) and isopropanol-ammonia-water (7: 1: 2 ), match Rf with the control sample. UV spectrum (ethanol): I max 269 nm (11600) and 234 nm (23000); And min 255 nm (10,000). The Dmtrrpynn content, determined at A max 500 nm (72000) in ethanol-60% HCNH mixture, is 99%.

Example 2. Synthesis of 5 -0-dimethoxytrityl-imidine-3-hydrophosphonate in the presence of quinoline. A suspension of Dmtr T (0.544 g, 1 mmol) in acetonitrile (4 ml) and quinoline (0.35 ml, 3 mmol) is treated (0.16

ml, 1.8 mmol) at -10 ° C with stirring until the precipitate dissolves within 10 minutes. Next, the reaction mixture was treated as described in Example 1. to obtain 0.65 g (92%) of b -O-dimethoxytritylthymidine-3-hydrophosphonate in the form of a triethylammonium salt with the same characteristics.

Example 3. Synthesis of 5 -0-dimethoxytritylthymidine-3-hydrophosphonate in the presence of 2,4,4-collidine. A suspension of Dmtr T (0.0544 g, 0.1 mmol) in acetonitrile (0.4 ml) and 2,4,6-collidine (0.039 ml, 0.3 mmol) is treated at 0 ° C and stirring PC1s (0.016 ml, 0 , 18 mmol) for 4 min. The thick suspension (2,4,6-collidine hydrochloride precipitates out) is treated as described in Example 1 to yield 0.063 g (90%) of the triethylammonium salt of the desired product.

Thus, the method allows to increase the yield of the target product by 6% (from 86 to 92%); simplify the process by eliminating the step of obtaining the tririmidazolylphosphine intermediate product and facilitating the purification process of the target product; speed up the process 9 times: reduce the cost of the target product.

Claims (2)

Claim 1. A method for preparing 5-0: dimethoxytritylthymidine-3-hydrophosphonate by treating 5 -0-dimethoxytritylthymidine in acetonitrile phosphorus trichloride in the presence of an amine at a low temperature, hydrolysis of the reaction mixture and isolation of the target product, characterized in that, in order to simplify and speed up the process, Also, increasing the yield of the target product, the suspension of 5 -0-dimethoxytritylthymidine is treated with phosphorus trichloride in the presence of a lower basic tertiary amine with a pKa lower. 2. The method according to claim 1, wherein the low basic tertiary amine is selected from the group of pyridine, quinoline, 2,6-lutidine, 2,4,6-collidine, N. N-dimethylaniline.