Synthesis of Adenosine-5
-O-(1-thio

Synthesis of Adenosine-5
-O-(1-thiotriphosphate). a) Enzymatic Synthesis. A mixture containing AMPS
(Na
salt; 56.7 mg, 0.14 mmol) and the phosphorylating extract (6 ml) was incubated at 28 for 8 h with gentle
stirring. The reaction was stopped by boiling for 3 min with subsequent clarification of the mixture by
centrifugation (5000  g, 10 min). The supernatant was chromatographed on a DEAE Sephadex A-25 (HCO 3
form, 1.8  27 cm) column with a linear gradient (0.001  1.0
, p H 7.6; 2  400 ml) of TEAB buffer. In order of
elution, the following products were isolated and transformed to their Na
salts: AMP (4 mg, 8%), AMPS
(13 mg, 23%), ATP (9 mg, 11%; HPLC: tR 3.67 min (max 258 nm)), and (Sp)-ATP
S (30.5 mg, HPLC: tR
4.21 min (max 258 nm); 37%; 48% based on the consumed AMPS).