WO1993023415A1

WO1993023415A1 - CHEMICAL SYNTHESIS OF 2',3'-DIDEOXYNUCLEOSIDE 5'-(α-THIO) TRIPHOSPHATES

Info

Publication number: WO1993023415A1
Application number: PCT/US1993/004290
Authority: WO
Inventors: Jasenka Matulic-Adamic; Leonid Beigelman
Original assignee: United States Biochemical Corporation
Priority date: 1992-05-13
Filing date: 1993-05-06
Publication date: 1993-11-25
Also published as: AU4236693A

Abstract

Method for synthesis of ddNTPαS by reacting a 2',3'-dideoxynucleoside with a mixture of thiophosphorylchloride and pyridine at a temperature less than 130 °C.

Description

DESCRIPTION

CHEMICAL SYNTHESIS OF 2',3'-DIDEOXYNUCLEOSIDE 5'-(α-THIO)TRIPHOSPHATES

Background of the Invention This invention relates to methods for the chemical synthesis of 2' ,3'-dideoxynucleoside 5'-(α- thio)triphosphates (ddNTPαs) . Bogachev, 13 Bioorg. Khim. 1683, 1987, describes synthesis of 2'-deoxynucleoside 5'-(α-thio)triphosphates. A mixture of triethylphosphate and trimethylphosphate was used to dissolve the starting nucleosides, and acetonitrile was used for preparation of the pyrophosphate solution. Auer et al., 8 Nucleosides & Nucleotides 849, 1989, describe synthesis of 2' ,3'-dideoxynucleoside 5'-0- (α-thio)triphosphates. Chu et al., 54 J. Orσ. Chem. 2217, 1989, describe a general synthetic method for 2',3'- dideoxynucleosides and 2 ' , 3 ' -didehydro-2 ' , 3 ' - dideoxynucleosides. Ludwig and Eckstein, 54 J. Org. Chem. 631, 1989, describe the chemical synthesis of nucleoside thiotriphosphates and cyclophosphorothioates using a chlorobenzodioxaphosphorinone reagent and sulfur.

Summary of the Invention This invention features a procedure for synthesis of ddNTPαS based on the pyridine catalyzed 5'-O-thio- phosphorylation of unprotected 2',3'-dideoxynucleosides. It allows synthesis of ddNTPαS's without purification of intermediate products. The method allows the chemical synthesis of ddNTPαS containing thymine, cytosine, adenine and guanine nucleotide bases and their purification, and thus provides nucleotide analogs suitable for DNA sequencing and related techniques.

The invention features methods which make use of triethylphosphate-trimethylphosphate mixtures as reaction solvents for dissolution of sensitive nucleosides at relatively low temperatures (below 130°C) . This low temperature prevents contamination of the reaction products with solvent-reacted groups. For example, C=N bonds present in the reactants are less likely to be cleaved under these conditions. The method also involves phosphorylating using acetonitrile with a tri-n- butylammonium pyrophosphate or tri-n-octylammonium pyrophosphate solution^". This step is advantageous over use of dimethylformamide.

The method of this invention substantially improves the yields of ddNTPαS over other methods, and (because intermediates are not isolated) end products can be obtained in a short time. This method is especially suitable for the preparation of radiolabeled compounds, and can be scaled up for ultigram synthesis. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The drawing will first briefly be described. Drawin :

The figure is a diagrammatic representation of a method of this invention for synthesis of a ddNTPαS. Synthesis:

Referring to the figure, in general the method of this invention features reaction of a nucleoside with a thiophosphorylating agent, e.g., PSC1₃ in pyridine, under conditions which allow dissolution to occur at a temperature below 130°C, e.g. , by use of a mixture of triethylphosphate and trimethylphosphate to dissolve the nucleoside. The resulting chemical is then converted to a ddNTPαS by reaction with a pyrophosphate dissolved in acetonitrile. Such a process does not require use of extremely dry solvents and yet provides high yields.

The following is an example of a method for synthesis and purification of ddNTPαS. This example is not limiting to the invention, and those of ordinary skill in the art will recognize that other equivalent methods can be used to produce the desired chemicals. Example;

2',3'-dideoxynucleoside (5 mmole) was dissolved in triethylphosphate or triethylphosphate- tri ethylphosphate 1:1, if necessary by heating at 120- 125°C for 4-6 minutes. The solution was quickly cooled to 0°C and thiophosphorylchloride (2 equivalents (eq) ) and pyridine (2 eq) added. The heterogenous mixture was stirred at 0-4°C 1-5 hours, then tributylamine (3 eq) and 0.25 M solution of tri-n-butylammonium pyrophosphate (4 eq) in acetonitrile added. The solution was stirred at room temperature (i.e. , about 24°C) for 10 minutes and quenched with 1 M triethylammonium bicarbonate (TEAB; water or aqueous ammonium hydroxide can also be used) buffer (10 ml/mmole) . The solution was set aside at room temperature for 24 hours and then concentrated to a syrup at 30°C. The residue was coevaporated two times with methanol and dissolved in 0.05 M TEAB (pH 7.5, 100 ml/mmole) . Purification:

The above solution was applied on the column of DEAE SephadexA-25 (HC0₃ ^~form) , and chromatography performed with a linear gradient of 2 L each 0.05 M and 1 M TEAB (pH 7.5), flow rate 2 ml/min, fraction size 25 ml. Fractions containing triphosphates were combined and concentrated in vacuo at 30°C. This solution was applied on the preparative C18 HPLC column and eluted with 100 mM TEAB

(pH 6.7) containing a linear gradient of acetonitrile from 0% to 20% in 25 minutes) . Sp and Rp isomers were collected together, acetonitrile was removed .in vacuo at 30°C, and the residue solution lyophilized. The resulting solid was dissolved in water and lyophilized, and this procedure repeated once more. The residue was dissolved in methanol and treated with the solution of sodium perchlorate in acetone. The precipitate was collected by centrifugation, washed 3 times with acetone, dissolved in water, and lyophilized to give 2'3'-dideoxynucleoside 5'- 0-(α-thio)triphosphates (sodium salts) as white solids in 65 - 88% yield.

Other embodiments are within the following claims.

Claims

1. Method for synthesis of a ddNTPαS comprising the step of dissolving a 2',3'-dideoxynucleoside with a mixture of thiophosphorylchloride and pyridine at a temperature less than 130°C.

2. Method for synthesis of a ddNTPαS comprising the step of reacting a 2' ,3'-dideoxynucleoside with a mixture of thiophosphorylchloride and pyridine in a solvent comprising triethylphosphate to form said ddNTPαS.

3. The method of claim 2 wherein said solvent further comprises trimethylphosphate.

4. The method of claim 3 wherein said triethylphosphate and said trimethylphosphate are provided in a 1:1 ratio.

5. The method of claim 2, 3 or 4 wherein said dideoxynucleoside and said solvent are heated at 120°C to 125°C.

6. The method of claim 1 wherein said agent is dissolved in triethylphosphate or triethylphosphate and trimethylphosphate.

7. The method of claim 1 wherein said method comprises a step of contacting tri-n-butylammonium pyrophosphate or tri-n-octylammonium pyrophosphate in acetonitrile with an intermediate in the synthesis of said ddNTPαS from said 2' ,3'-dideoxynucleoside.