DD212391A3 - METHOD OF GENERATING (GAMMA-HIGH 32 P) ADENOSINE-5'-TRIPHOSPHATE HIGH SPECIFIC ACTIVITY - Google Patents

Abstract

Process for the preparation of (gamma-high 32 P) adenosine-5'-triphosphate (ATP) with high specific activity of the formula I. The object of the invention is to develop a technically simple, fast process which gives good yields. According to the invention, a small batch volume of adenosine-5'-diphosphate is enzymatically phosphorylated on the molar scale under certain concentration ratios of corresponding substrates (gamma-high 32 P) ATP. After a short reaction time, the product can be isolated by column chromatography in a simple procedure.

Description

Dr. E. Bauschke R. Berger ZfK M. Kiessling ZfK H. Kreißl

"Method for producing / J- P / adenosine 5'-triphosphate of high specific activity"

Field of the Invention The invention relates to a verj adenosine 5'-triphosphate (ATP) of the formula I.

The invention relates to a method for P-labeling of

0 0 0 _θ Ο-32_Ι_ _Ο -! _ Θ- | -Ο

M ⁺ = H ⁺ or other cations.

Fields of application are radiochemical synthesis and nucleotide chemistry.

Characteristic of the known technical solutions

A number of methods are known in the literature which allow to obtain Q- P _- / ATP.

According to Waiseth (TP Walseth, R, A. Johnson Biochem et Biophys., Acta 5 p., (1979) 11-31), reaction of ³² P-phosphate with D-glyceraldehyde-3-phosphate, which was prepared by triose isomerase from dihydrosyacetone phosphate, produced the desired 1,3-diphosphoglycerate, which itself enzymatically reacts with adenosine 5'-diphosphate (ADP).

»Hi» αλλ a. . ι \ er ε: ι · * - *

232854 7

In. this. Approach is used, a very complex enzyme mixture. Enzymes are very sensitive to ß-radiation of higher intensity, so Walseth, as upper radiochemical

TO-

Concentration: 10 mCi H, P0 "/ 50 All reaction volumes". : '. ,

A much simpler method according to Gilbert, Mazam (W. Gilbert, A.M.Maxam, Laboratory Communication, and I.M. Glynn, J. "B. Chappell. Biochem.,"<T. £ 0 .1964) '·; 147 - "· - 149) does not result in such a high specific stop activity ^1,...

For synthesis; of Zp-P / ATP: lower specific activity ;, a method of 'R.' Gibbs, E.L / L. Röddy, E .. Titus ¹ CJ * -

Biol. Chem. 240 (1965) 2181). "A synthetic prescription for which high specific activity has not been described, however, has been:

. Aim DER invention · .. '''''The Erfindung- has: the ^one goal ,, technically, möglichst- simple method:. ,, develop the production DIE: to from, nmol amounts -. hochmarkier specific system FFI XJ ATP by the action of a complex enzyme mixture in a short time ensured ......

Presentation of the essence of the invention : According to the invention are compounds of the formula. X prepared by: man: in a one-pot process, the:

32 "

amount of carrier-free H-, PO ,, which: has been previously lyophilized, in one. Substrate mixture receives .. The reaction is started specifically, purified enzymes by the addition and after a few. Minutes z. B '.. stopped by ethanol ... Hach lyophilization and recovery in. Some water, the reaction mixture on. an anion exchange column, e.g. B .. DEAE-Sephadex: A 25V: added and the desired product isolated by elution - E ^ ne ^ High performance liquid chromatography separation at reversed phase is also possible.

UHUJ

embodiment

The following embodiment explains the invention in more detail. ,

925 MBg. carrier-free E ₃ ³² PO ₄ CROTOP-H ₃ ³² PQ ₄ , ZfK-Rossendorf) were in a. Plastic tubes. lyophilized »The lyophilisate was in. 80 All. of a substrate mixture containing 60% of TRIS / EC1; pH = 8; 0.6.mffi of nicotinamide adenine dinucleotide; 0.37 mM. ADP ;. 0, -58mM Fructose-1, 6-diphosphate 1.5 x NaM an, MgCl2; 1.3 with at Chelaplex III was ·. In addition were. TOO; / Ug- of the egg mixture of aldolase, phosphoglycerate kinase., Glyceraldehyde phosphate dehydrogenase (mass ratio 2: · .1 ': 2) in.-1'O; / Ul 0, ₁ M TRIS / HCl Puff he ,. The mixture was mixed for 5 minutes with 300 yl of absolute ethanol. The Df * 2j ATP yield was greater than 80% after lyophilization, and uptake of the residue in 0.55 ml of H ₂ O is applied to a DEAE-Sephadex A 25 column in (HCO ₃ ") 'form and mixed with. continuous gradient TOO ml HpO. 100 ml of T K triethylammonium bicarbonate solution eluted ... Dia. Dimension, the column was 1 χ 10 cm.

Between; 110: -120 ml, a fraction was collected which contained the '' ΈΖ &. ' With a radiochemical purity of. 98 % contained .. ·;

The product characterization was carried out by thin-layer chromatography. inactive ATP as a comparison.

Rrv = 0.28 - on "polyethyleneimine cellulose precoat plates of Fa.

Merck. '

Eluent: 0.5 MKH ₂ PO ₄ , pH = 3.5

Claims (1)

L JZ834 J 32 _r Process for the preparation of / γ- P / ATP of high specificity Activity of the formula O O 32 "t jO- Π ³ -OPOP-0 k% o * 4M M ⁺ = H ⁺ or other cations characterized in that adenosine 5'-diphosphate is present in a substrate mixture consisting of TRIS / HCl; Nicotinamide adenine dinucleotide, r ADP ^ fructose-diphosphate, MgCl ₀ and chela- 32
plex ΙΙΓ, ί with unsupported H ₃ PO ₄ within 2 - 15 minutes through an enzyme cycle. consisting of aldolase, phosphoglycerate kinasei; Glyceraldehyde phosphate dehydrogenase becomes phosphorylated enzymatically *