Biochimica et Biophysica Acta, 331 (1973) 307-309 ~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 97857

A CHEMICAL SYNTHESIS OF ADENOSINE 5'-[y-32p]TRIPHOSPHATE

SIDNEY M. I-IECHT and JOHN W. KOZARICH

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.)

(Received July 3rd, 1973)

SUMMARY

Adenosine 5'-[y-32p]triphosphate has been prepared from adenosine 5'-diphos- phate by condensation of the phosphorimidazolidate of ADP with 32p i. No purifica- tion of intermediates is required in this efficient synthesis, which is convenient for micro-scale conversions. This procedure permits the preparation of [y-32p]ATP of high specific activity and should be applicable to the synthesis of many nucleoside triphosphates, including some which may not be accessible by enzymatic means.

The preparation of adenosine 5'-[y-a2p]triphosphate has been achieved enzy- matically by the action of glyceraldehyde-3-phosphate dehydrogenase and 3-phospho- glyceric acid kinase on adenosine 5'-diphosphate 1-3. A chemical procedure has also been reported for the synthesis of [y-a2p]ATP 4'5 although the method has been indi- cated 6 not to be convenient on a small scale. This report is concerned with an additio- nal chemical procedure for the preparation of [y-32p]ATP based on the activation of adenosine 5'-diphosphate with 1, l'-carbonyldiimidazole (Kozarich, J. W., Chinault, A. C. and Hecht, S. M., unpublished), by extension of the work of Hoard and Ott 6, and Cramer eta/. 7-1°. The resulting phosphorimidazolidate is then treated with 32p i to afford [V-32p]ATP.

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308 S .M. HECHT, J. W. KOZARICI-[

In a typical experiment, adenosine 5'-diphosphate (5/~moles), as the anhydrous mono tri-n-butylammonium salt 6, in 100 pl of N,N-dimethylformamide was treated with 25 pmoles of I, l'-carbonyldiimidazole in 50 pl of N,N-dimethylformamide. The resulting solution was maintained overnight under anhydrous conditions and then treated with 40 #moles of methanol for 30 min at room temperature. The solution was then treated with tri-n-butylammonium phosphate (1 or 25 pmoles) (spec. act. 0.1 Ci/mole) in 100 #1 of N,N-dimethylformamide and the reaction mixture was maintained at room temperature for 36 h. The solvent was removed by evaporation under diminished pressure and the product was purified by chromatography on DEAE -cellulose (2 cm × 25 cm), elution with a linear gradient of NH4HCO 3 (11 total volume; 0-0.5 M; 15-ml fractions) at a rate of 150 ml/h. The appropriate fractions were pooled and desalted by repeated evaporations of portions of water at 45 °C under diminished pressure. The reaction was carried out with either the phosphorimidazolidate or tributyl- ammonium phosphate in 5-fold excess relative to the other, to conserve isotope or maximize ATP production, respectively. In either case the yield of [7-32p]ATP was approx. 60 %, based on the limiting reagent. The remaining unreacted material was recovered as adenosine 5'-diphosphate and inorganic phosphate. The synthetic ATP had the same specific activity as the starting phosphate, corrected for radioactive decay.

To determine the distribution of 32p in the synthetic ATP, the triphosphate was utilized as a source of phosphate in the hexokinase-catalyzed formation of glucose 6-phosphate from glucose. Fig. 1A depicts the radioactivity profile of a sample of [7-32p]ATP, after elution with 0.52 M KH2PO 4 (pH 3.5) on a polyethyleneimine thin-layer chromatography plate ~1. The arrow depicts the position of an authentic sample of ATP (Rp = 0.16). Fig. 1B shows the profile of the ATP after utilization

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Fig. 1. Chromatography on thin-layer plates (Brinkmann MN polygram cel 300 PEI; 0.52 M KH2PO4, pH 3.5) of [~,-32P]ATP before (A) and after (B) treatment with hexokinase for 6 h (0.I M Tris-HC1, pH 8.0, 6.2 mM MgCI2, 1 M glucose, 25 pM [~,-a2p]ATP, 0.04 unit hexokinase) 12.

CHEMICAL SYNTHESIS OF [7-32P]ATP 309

in the hexokinase assay. All o f the detectable rad ioac t iv i ty was associa ted with glucose 6-phospha te (Rp = 0.66). N o rad ioac t iv i ty was observed co r respond ing to the known RF values o f A T P or A D P (arrow, R r = 0.32). This p rocedure affords a me thod for the p repa ra t ion o f a variety o f nucleoside 5 '-[V-32p]triphosphates, including some which may no t be accessible by enzymat ic means. Because no purif icat ion o f inter- media tes is necessary, and the t r ans fo rma t ion can be effected convenient ly on a micro- scale, the p rocedure may represent an a t t ract ive a l ternat ive to enzymat ic methods.

ACKNOWLEDGMENT

Acknowledgmen t is made to the donors of the Pe t ro leum Research Fund , admin i s t e red by the A.C.S. , for f inancial suppor t of this research.

REFERENCES

1 Penefsky, H. S. (1967) Methods Enzymol. 10, 702-703 2 Post, R. L. and Sen, A. K. (1967) Methods Enzymol. 10, 773-776 3 Glynn, I. M. and Chappel, J. B. (1964) Biochem. J. 90, 147-149 4 Wehrli, W. E., Verheyden, D. L. M. and Moffatt, J. G. (1965) J. Am. Chem. Soc. 87, 2265-2277 5 Moffatt, J. G. (1967) Methods Enzymol. 12, 182-192 6 Hoard, D. E. and Ott, D. G. (1965) J. Am. Chem. Soc. 87, 1785-1788 7 Cramer, F., Schaller, H. and Staab, H. A. (1961) Chem. Ber. 94, 1612-1621 8 Schaller, H., Staab, H. A. and Cramer, F. (1961) Chem. Ber. 94, 1621-1633 9 Crarner, F. and Schaller, H. (1961) Chem. Ber. 94, 1634-1640

I0 Cramer, F. and Neunhoeffer, H. (1962) Chem. Ber. 95, 1664-1669 11 Walsh, Jr, C. T. and Spector, L. B. (1971) J. Biol. Chem. 246, 1255-1261 12 Secrist, III, J. A., Barrio, J. R., Leonard, N. J. and Weber, G. (1972) Biochemistry 11, 3499-3506