DD213223A1 - PROCESS FOR PREPARING / ALPHA HIGH 32 P / NUCLEOSIDE 5'-PHOSPHATES OF HIGH SPECIFIC ACTIVITY - Google Patents

Abstract

Process for making & a-32P! Nucleoside-5-phosphates with high specific activity of the formula I. The object of the invention is to develop a technically simple, rapid process which, with good yields, gives products with maximum specific activity. According to the invention, in a small total volume, carrier-free H332PO4 is reacted with nucleosides in a rapid chemical synthesis in an organic solvent. Without intermediate purification, the product of the first stage of the synthesis is then further enzymatically phosphorylated in the aqueous buffer system. The product is isolated by column chromatography in a simple procedure.

Description

-A-

Dr. t. Bauschke

H. Kreißl

A. Miinzenberg

32 "Process for Making / c ^ - P / Nucleoside 5 ¹ Phosphate

high specific activity "

Field of application of the invention

The invention relates to a method for the £ cL- P_7 labeling of ribonucleoside and deoxyribonucleoside 5'-phosphates of the formula I.

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

R. = OH R _rt = OH or H

V /

pi D ^ R = HR ₂ = OH or H

X = phosphate »diphosphate» triphosphate

Field of application is the radiochemical synthesis, nucleotide chemistry.

Characteristics of Known Technological Solutions A number of methods are known in the literature which allow one to obtain / δC-P_7-labeled nucleotides of high specific activity. The best known methods described by TF Walseth, R. A · Johnson and AE Reeve, respectively and

R. C. Huang, are fully-gastric 4-step reactions * with

32 träg.erfreiera Pi is added to ADP in an enzymatic synthesis

32 Τ "" Ρ / ΑΤΗ reacted, which under the action of T4 polynucleotide kinase phosphorylated 3 * XMP at the 5 'position. Pl nuclease cleaves the 3'-phosphate to give 5 * / ^ P / XMP _f

which in turn is phosphorylated by a kinase mixture zura / cC - PjXTP «

The disadvantage of this process is the use of various highly purified enzymes and substrates for the synthesis of the P32-labeled nucleoside monophosphate. The specific activity of the final product is below the specific activity of the intermediate / f- P / A7P. Syraon described in 1978 a simpler chera-enzymatic process using a mixture of labeled and unlabeled H ₃ PO ₄ in the 1 x step which did not meet the requirements of a process in terms of yields and, above all, in the specific activities of the products.

Object of the invention

The aim of the invention is to develop a method which is as simple as possible in terms of technology * that is the production of nmol quantities

32 to specifically highly labeled ^ bC- pyNucleosidphosphaten in short reaction times and allows good yields.

Explanation of the essence of the invention

According to the invention, compounds of the formula I are prepared by reacting in a one-pot process the corresponding activity.

amount of carrier-free H ₃ PO 4, which was lyophilized, in a chemical reaction with addition of a Koraplexbildners «z. B · Fe (III) -EDT is reacted with the protected or unprotected nucleoside under the action of trichloroacetonitrile / triethylamine in the optimum ratio to the nucleoside 5 ^f -monophosphate. · After 30-60 min., The reaction mixture is poured into Tris buffer with increased Mg- Added content ^ after addition of substrates and corresponding kinases, the / P ./nucleoside-S'-raonophosphate is converted to / © C-P / nucleoside 5 * -triphosphate within 2-8 h «

The / xL- P / nucleoside S'-triphosphate is separated from the reaction residue by column chromatography on an anion exchanger, eg. 3. OEAE-Sephadex A25 _f "Elution takes place after a single gradient gradient."

The following examples illustrate the connection in more detail:

EMBODIMENTS 1. Example

% Q rnCi carrier-free Η-, ΡΟ. from the ZfK Rossendorf were lyophilized with 0.5 .umol Chelaplex II in a small tube * The lyophilisate was in 50 .ul dry dimethyl sulfoxide, the 5 rag 2-Oesoxyadenosin contained »added. The reaction was started by adding 5 μl of triethylamine and 10 μl of a 20% trichloroacetonitrile DMSO solution. After 60 minutes the Reaktionsgetnisch was Rait 0.5 ml of a 50 mM Tris-HCl buffer, pH = 8 _r l, offset, the 20 TNM KCl, 30 mM MgAc _2, 5 önd .ul Hercaptoethanol, 5 nmol of ATP, 5 , umol PEP contained * Mach addition of SO, ug myokinase and 100 .ug Pyruvatkin _a se the reaction is started, and after the ER-

from

rich in a yield of Nucleosidtrxphosphat 50 - 70 % , the Reaktionsgeraisch was separated by column chromatography on DEAE-Sephadex-A25 in bicarbonate form. The reaction mixture was added to a 0.5 χ 5 era column and washed with 20 ml of water, then with 20 ml of 0.3 M Triäthylaramoniunibicarbonat «Subsequently, in a continuous gradient 25 ml of 0.3 M against 25 ml of 0.7 M Triäthylaramoniumbicarbonat with an elution rate of 1 ml / min, the desired triphosphate after ca, 10 ml as a 5-7 ml fraction isolated. The radiochemical purity of the fraction was 99 %

After lyophilization and incorporation of ^ C-dATP in 50 % ethanol, the absolute yield of product activity was 28 %. "

The product characterization is carried out by thin-layer chromatography with inactive dATP comparison

Rp = 0.31, on PEI-cellulose from Merck mobile phase 0.5 M KH ₃ PO ₄ -Puffer pH = 3.5

2 »Example

32 10 μl of carrier-free H ₃ PQ ₄ in 60 μl 0.1 N hydrochloric acid from the ZfK Rossendorf were mixed with 10 μl (1 μmol) of a chelaplex II solution and lyophilized in a small plate tube. The lyophilizate was taken up in 50 μl of dried dimethylsulfoxide containing 5.0% of 2 * -acetyl-3'-deoxyadenosine *. The reaction was started by adding 5 μl of triethylamine and 10 μl of a 20% trichloroacetonitrile / DHSQ. Solution started »After 60 minutes, the reaction mixture was treated with 200 μl of 25 % NH ₄ OH and left at room temperature for 2 hours. After lyophilization of the mixture, the residue was dissolved in 0.5 μl of 50 mM Tris-HCl buffer, pH = 8.1 , which was 20 mM in KCl , 30 mH in MgAc ₂ and contained 5, μl of mercaptoethanol, 5 nmol of ATP, 5 μmol of phosphoenolpyruvate. Upon addition of 50 μg of myokinase and 100 μg of pyruvate kinase, the reaction was started and subjected to column chromatography on DEAE-Sephadex A25 after reaching a yield of nucleoside 5 ^1- triphosphate of 72 % after 4 hours.

For this purpose, the reaction mixture was added to a 0.5 × 5 cm column, washed with 20 μl of HpO and then with 20 ml of 0.3 M triethylammonium bicarbonate. Subsequently, in a continuous gradient of 25 μl of 0.3 M against 25 ml of 0.7 M triethylammonium bicarbonate, the desired triphosphate was isolated after about 10 ml as 8 ml fraction at an elution rate of 1 ml / min. The radiochemical purity of the fraction was 99% / ct ~ ² · P7-3'dATP

32 After lyophilization and incorporation of the / X-P7-3'dATP in 50% ethanol, the absolute yield of product activity was 26

The product characterization is carried out by thin-layer chromatography with inactive substance comparison »Rp = 0.28 on PEI-cellulose from Merck eluent 0.5 M KH ₃ PO ₄ -Puffer, pH = 3.5

Claims (1)

-i- invention claim
Procedure for Hers'
high specific activity of the formula I ₁ 32 Process for the preparation of / oL-P7 nucleoside 5'-phosphates in the B = adenine, thyrain, guanine, cytosine, uracil and their derivatives
R ₁ ^ OH or H
R ₂ = OH or H X = phosphate »diphosphate, triphosphate 32
mean ^ from carrier-free H ₃ PO. and nucleosides by a stepwise chemical enzymatic synthesis », characterized in that carrier-free H ₃ PO. with the addition of a complexing agent, z * B *. Fe (III) -EDT lyophilized and reacted with a protected or unprotected nucleoside with the addition of trichloroacetonitrile and triethylamine to the nucleoside 5'-monophosphate, then added to Tris buffer with increased magnesium content and after addition of appropriate 32
Kinases after 2 - 8 h / cC- P? Nticleoside-5'-triphosphate, is purified by column chromatography.