- Email: [email protected]
A chemical synthesis of adenosine 5′-[γ-32P]triphosphate
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'-diphosphate by condensation of the phosphorimidazolidate of ADP with 32pi. No purification 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 enzymatically by the action of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglyceric 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 indicated 6 not to be convenient on a small scale. This report is concerned with an additional 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of adenosine 5'-diphosphate with 1, l'-carbonyldiimidazole (Kozarich, J. W., Chinault, A. C. and Hecht, S. M., unpublished), by extension of the work of Hoard and Ott 6, and Cramer eta/. 7-1°.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. .NHz
NHz
oo ,
o-io!o.
0-0"
c
^
o
oo.
,
~
>
"
HO OH NH;,
000 ~ . O~2~O~OPOH2C n
I
HO OH
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 tributylammonium 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
? o x
i
v
o'.so RF
1 0.7s
Fig. 1. Chromatography on thin-layer plates (Brinkmann M N 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 m M MgCI2, 1 M glucose, 25 p M [~,-a2p]ATP, 0.04 unit hexokinase) 12.
CHEMICAL SYNTHESIS OF [7-32P]ATP
309
in the hexokinase assay. All o f the detectable r a d i o a c t i v i t y was associated with glucose 6 - p h o s p h a t e (Rp = 0.66). N o r a d i o a c t i v i t y was o b s e r v e d c o r r e s p o n d i n g to the k n o w n RF values o f A T P or A D P (arrow, R r = 0.32). This p r o c e d u r e affords a m e t h o d for the p r e p a r a t i o n o f a variety o f nucleoside 5'-[V-32p]triphosphates, including some which m a y n o t be accessible by e n z y m a t i c means. Because no purification o f intermediates is necessary, a n d the t r a n s f o r m a t i o n can be effected conveniently on a microscale, the p r o c e d u r e m a y represent an attractive alternative to enzymatic methods. ACKNOWLEDGMENT A c k n o w l e d g m e n t is m a d e to the d o n o r s o f the P e t r o l e u m R e s e a r c h F u n d , a d m i n i s t e r e d by the A.C.S., for financial s u p p o r t o f this research.
REFERENCES 1 2 3 4 5 6 7 8 9 I0 11 12
Penefsky, H. S. (1967) Methods Enzymol. 10, 702-703 Post, R. L. and Sen, A. K. (1967) Methods Enzymol. 10, 773-776 Glynn, I. M. and Chappel, J. B. (1964) Biochem. J. 90, 147-149 Wehrli, W. E., Verheyden, D. L. M. and Moffatt, J. G. (1965) J. Am. Chem. Soc. 87, 2265-2277 Moffatt, J. G. (1967) Methods Enzymol. 12, 182-192 Hoard, D. E. and Ott, D. G. (1965) J. Am. Chem. Soc. 87, 1785-1788 Cramer, F., Schaller, H. and Staab, H. A. (1961) Chem. Ber. 94, 1612-1621 Schaller, H., Staab, H. A. and Cramer, F. (1961) Chem. Ber. 94, 1621-1633 Crarner, F. and Schaller, H. (1961) Chem. Ber. 94, 1634-1640 Cramer, F. and Neunhoeffer, H. (1962) Chem. Ber. 95, 1664-1669 Walsh, Jr, C. T. and Spector, L. B. (1971) J. Biol. Chem. 246, 1255-1261 Secrist, III, J. A., Barrio, J. R., Leonard, N. J. and Weber, G. (1972) Biochemistry 11, 3499-3506
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'-diphosphate by condensation of the phosphorimidazolidate of ADP with 32pi. No purification 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 enzymatically by the action of glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglyceric 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 indicated 6 not to be convenient on a small scale. This report is concerned with an additional 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. .NHz
NHz
oo ,
o-io!o.
0-0"
c
^
o
oo.
,
~
>
"
HO OH NH;,
000 ~ . O~2~O~OPOH2C n
I
HO OH
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 tributylammonium 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
? o x
i
v
o'.so RF
1 0.7s
Fig. 1. Chromatography on thin-layer plates (Brinkmann M N 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 m M MgCI2, 1 M glucose, 25 p M [~,-a2p]ATP, 0.04 unit hexokinase) 12.
CHEMICAL SYNTHESIS OF [7-32P]ATP
309
in the hexokinase assay. All o f the detectable r a d i o a c t i v i t y was associated with glucose 6 - p h o s p h a t e (Rp = 0.66). N o r a d i o a c t i v i t y was o b s e r v e d c o r r e s p o n d i n g to the k n o w n RF values o f A T P or A D P (arrow, R r = 0.32). This p r o c e d u r e affords a m e t h o d for the p r e p a r a t i o n o f a variety o f nucleoside 5'-[V-32p]triphosphates, including some which m a y n o t be accessible by e n z y m a t i c means. Because no purification o f intermediates is necessary, a n d the t r a n s f o r m a t i o n can be effected conveniently on a microscale, the p r o c e d u r e m a y represent an attractive alternative to enzymatic methods. ACKNOWLEDGMENT A c k n o w l e d g m e n t is m a d e to the d o n o r s o f the P e t r o l e u m R e s e a r c h F u n d , a d m i n i s t e r e d by the A.C.S., for financial s u p p o r t o f this research.
REFERENCES 1 2 3 4 5 6 7 8 9 I0 11 12
Penefsky, H. S. (1967) Methods Enzymol. 10, 702-703 Post, R. L. and Sen, A. K. (1967) Methods Enzymol. 10, 773-776 Glynn, I. M. and Chappel, J. B. (1964) Biochem. J. 90, 147-149 Wehrli, W. E., Verheyden, D. L. M. and Moffatt, J. G. (1965) J. Am. Chem. Soc. 87, 2265-2277 Moffatt, J. G. (1967) Methods Enzymol. 12, 182-192 Hoard, D. E. and Ott, D. G. (1965) J. Am. Chem. Soc. 87, 1785-1788 Cramer, F., Schaller, H. and Staab, H. A. (1961) Chem. Ber. 94, 1612-1621 Schaller, H., Staab, H. A. and Cramer, F. (1961) Chem. Ber. 94, 1621-1633 Crarner, F. and Schaller, H. (1961) Chem. Ber. 94, 1634-1640 Cramer, F. and Neunhoeffer, H. (1962) Chem. Ber. 95, 1664-1669 Walsh, Jr, C. T. and Spector, L. B. (1971) J. Biol. Chem. 246, 1255-1261 Secrist, III, J. A., Barrio, J. R., Leonard, N. J. and Weber, G. (1972) Biochemistry 11, 3499-3506