DD293831A5 - PROCESS FOR PREPARING NUCLEOSIDE 5'-O- (1- [EXP35S] THIOPHOSPHATES) HIGH SPECIFIC ACTIVITY - Google Patents

Abstract

The invention relates to a process for the preparation of the formula I with the field of application of radiochemistry, nucleotide chemistry, nucleic acid labeling, nucleic acid research, biochemistry. According to the invention, N- or OH-protected nucleosides are reacted in excess with phosphinylating reagents in anhydrous organic solvents, and after purifying the product, the desired 35S-labeled * is produced with elemental 35S high specific activity as oxidizing agent, which may optionally be added with kinase after purification by column chromatography * is phosphorylated. Formula I {method; * Radiochemistry; nucleotide; nucleic acid label; Nucleinsaeureforschung; Biochemistry; elementary 35S; kinase}

Description

Field of application of the invention

The invention relates to a process for the preparation of ^3B S-labeled high specific radioactivity deoxyribonucleoside and ribonucleoside 5'-O- (1-thiophosphates) of general formula I.

X-Q-

B = adenine, thymine, guanine, cytosine, uracil and their derivatives

R ₁ = OH ₁ R ₂ = OH or H

R ₁ = H, R ₂ = OH or H

X = [ ³⁵ S] thiophosphate, 1- [ ³⁵ S] thiophenol phosphate.

Areas of application are nucleotide chemistry, nucleic acid labeling, nucleic acid research, radiochemistry, biochemistry.

Characterization of the known prior art

Known from the literature are chemical, chemical-enzymatic and enzymatic synthesis methods for nucleoside 5'-O- (1-thiophosphates) and - (1-thiopolyphosphate) and thiophorophosphate oligonucleotides, respectively.

Non-radioactive thiophosphate oligonucleotides were mostly prepared by the phosphoramidite synthesis method on the solid support followed by oxidation of the phosphorous diesters with elemental sulfur (Taylor, IW, Schmidt, W .; Corstick, R., Okruszek, A. Eckstein, F., Nucleic Acids Res. 13 [1985] 8749); nonradioactive nucleoside B'-O-ii-thiotriphosphates) were prepared by reaction with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one followed by hydrolysis (Ludwig, J .; Eckstein, F., J. Org. Chemistry 54 [1989] 631).

Syntheses ³⁵ S-labeled nucleoside 5'-O- (1-thiophosphates) are known to be either enzymatically derived from [ ³⁵ S] -thiophosphate via [ ³⁵ S] -ATP S (Eckstein, F. Biochem., Biophys 483 [1977] 1-5; Cassidy, PS; Kerrick, WGL, Biochim Biophys. Acta 565 [1979] 209) or by reacting nucleosides with P ³⁵ SCI ₃ , followed by hydrolysis and enzymatic phosphorylation with a kinase mixture (Bartlett, PA; Eckstein, F., J. Biological Chemistry 257 [1982] 8879). P ³⁵ SCI ₃ and [ ³⁵ S] thiophosphate are commercially available only in low specific radioactivity. Routes to specifically highly labeled nucleoside 5'-O- (1- [ ^3S S] -thiophosphates) and polyphosphates are not described.

Object of the invention

The aim of the invention is to develop a chemical process which is as simple as possible in the art and which enables the preparation of nmol amounts of specifically highly labeled nucleoside 5'-O- (1 - [ ^3S S] -thiophosphates which may then be further added to nucleoside 5'-O- (1- [ ³⁵ S] thiotriphosphates) can be reacted in good yields.

Explanation of the essence of the invention

According to the invention compounds of formula I are prepared by reacting N- and OH-protected nucleosides with Phosphinylierungsreagenzien, for example bis (diisopropylamino) - (2-cyanoethoxy) phosphine, in anhydrous organic solvents such as dioxane, pyridine or acetonitrile with O ₂ exclusion implements. There is no dinucleoside phosphite as by-product. After purification of the product by extraction, the ³⁵ S-labeling is carried out by elemental ^3S S-sulfur of high specific activity> 77 GBq / mA as oxidizing agent under N ₂ protective gas. On a DEAE-Sephadex A 25 exchange column, the product is purified and, after isolation, optionally with a kinase / substrate mixture, phosphorylated to nucleoside 5'-O- (1- [ ³⁵ S] -thiopolyphosphate). In a subsequent chromatographic purification on DEAE-Sephadex A25, the desired product is isolated. The inventive method is technically easy to carry out and leads to specifically highly labeled compounds

 The following exemplary embodiments explain the invention in more detail:

embodiments

1st example

Synthesis of Thymidine-5'-O- (1- [ ³⁵ S] thiotriphosphate)

2.8 mg (ΙΟμιηοΙ) of 3'-acetylthymidine were dissolved together with 2.4 mg (34μιηοΙ) tetrazole in 400μΙ dry dioxane. Under N ₂ atmosphere, 18 μΙ (60 mol) of bis (diisopropylamino) - (2-cyanoethoxy) phosphine was added. After 3 h the solution was concentrated, taken up in methylene chloride and washed twice with 500μΙ 500 μΙ 2% NaHCO ₃ solution and extracted once with 500 μΙ saturated NaCl solution. The organic phase was dried with Na ₂ SO ₄ , separated and concentrated. The residue was taken up in an N ₂ atmosphere with 70 μΙ (2 mCi / 1 μιτιοΙ) ³⁵ S / pyridine / CS ₂ / NEt ₃ (v: v: v: 1: 1: 0.1) and allowed to stand overnight, concentrated and treated with 100μΙ concentrated ammonia solution at room temperature for 1 h. The reaction mixture was purified on a continuous gradient DEAE-Sephadex A25 column (V = 5ml), 20ml 0.15M / 20ml 0.5M TEAB. After isolation, the product isolated in the first 10 ml was taken up in 500 .mu.l of 50 mM Tris-HCl buffer and mixed with 200 .mu.l of an enzyme-substrate mixture containing 20 mM KCl, 30 mM MgCl ₂ , 10 mM phosphoenolpyruvate, 10 mM ATP, 5 mM dithiothreitol and 800 μg of nucleoside monophosphate kinase from bovine liver and 100 μg pyruvate kinase from rabbit muscle. After 22 hours, thymidine-5'-0- (1- ^[35 S] was -thiotriphosphat formed with a related ³⁶ S sulfur radiochemical yield of 28%. The reaction mixture was added (on a DEAE-Sephadex A25 column V = 5ml) separated in bicarbonate form with a linear gradient of 20 ml each 0.15M TEAB / 0.7 M TEAB. [ ³⁵ S] TTPaS was isolated with a total yield of 19% at a buffer concentration of 0.5 M. The product characterization was carried out by thin-layer chromatography on silica gel from Merck. R _F = 0.16 (LM nPropanol: NH ₃ : H ₂ O: 6: 3: 1)

2nd example

Synthesis of 2'-deoxyadenosine-5'-O- (1- [ ³⁵ S] thiophosphate)

3,2mg (ΙΟμιτιοΙ) N-benzoyl-3'-acetyl-2'-deoxyadenosine and 2,3mg tetrazole were dissolved ϊηδΟΟμΙ dioxane and under N ₂ atmosphere with 18μΙ (ΘΟμιηοΙ) bis (diisopropylamino) - (2-cyanoethoxy) Phosphine added.

After 6 hours, the mixture was concentrated and the residue taken up in 500 μΙ methylene chloride. Two times with 500 μΙ each 2% NaHCO ₃ and once with 500 μΙ saturated NaCl was washed. The organic phase was dried over Na ₂ SO ₄ , separated and concentrated. The residue was ΓηΚ70μΙ a pyridine / CS ₂ / triethylamine solution (v: v: v, 1: 1: 0.1) containing 2mCi ³⁵ S (1 μηιοΙ), recorded and left overnight under N ₂ atmosphere.

After concentration, the residue was concentrated in 100μΙ concentrated. Ammonia, incubated for 1 hour and then separated via a DEAE-Sephadex A25 column (V = 5 ml) with continuous gradient 20 ml 0.05M TEAB / 20 ml 0.5M TEAB.

The desired product was eluted at about 0.2 M TEAB in an overall yield of 21%.

The product characterization was carried out by thin-layer chromatography on silica gel.

Rf = 0.4, eluent: nPropanol: NH ₃ : H ₂ O wie6: 3: 1.

Claims (3)

1. A process for the preparation of nucleoside 5'-O- (1 [ ³⁵ S] thiophosphates) high specific activity of the general formula I. X-O in the B = adenine, thymine, cytosine, uracil and their derivatives Ri = OH, R ₂ = OH or H or R ₁ = H, R ₂ = OH or H χ = [ ³⁵ S] -thiophosphate, 1- [ ³⁵ S] -Thiopoly phosphate mean, characterized in that in an anhydrous organic I solvent under Sau first off-exclusion OH- or N-protected or unprotected nucleosides with Phosphinylierungsreagenzien in excess, then the resulting product with elemental ³⁵ S-sulfur high specific activity> 77GBq / mA ³⁵ S-labeled, purified and optionally phosphorylated with a kinase / substrate mixture enzymatically. 2. The method according to claim 1, characterized in that is used as Phosphinylierungsreagenz bis (diisopropylamino) - (2-cyanoethoxy) phosphine. 3. The method according to claim 1, characterized in that the ³⁵ S-labeling is carried out under N ₂ protective gas.