Adenosine 3/,5'-cyclic phosphorothioate (cAMPS) was synthesized. Its interaction with cyclic nucleotide phosphodiesterase from beef heart and rabbit brain as well as with cAMP-dependent protein kinase from beef heart was studied and compared with that of cAMP. Both diastereomers of cAMPS are slowly hydrolyzed by the diesterases w'ith rates about '/so to Vi 50 that of cAMP. The hydrolysis of cAMP (ATm In an effort to modify the biological properties of adenosine 3/,5/-cyclic phosphate, a large number of analogs has been synthesized with alterations in the base, the sugar, and the phosphate part of the molecule (Simon et al.. 1973). In an attempt to synthesize cAMPS, one of us has reacted adenosine 5'-phosphorothioate with ', '-dimethylformamide dimethyl acetal and triisopropylbenzenesulfonyl chloride (Eckstein, 1970). Although certain aspects of the reaction could not be explained, such as the absence of any cAMP formation as well as the inability to detect the two expected diastereomers. the product was assumed to be cAMPS based on its electrophoretic mobility, its elementary analysis, and its chemical shift in the P nmr.This compound was, unexpectedly, neither substrate nor inhibitor for 3',5/-cyclic nucleotide phosphodiesterases from various sources (Eckstein and Bar, 1969). Because of these discrepancies it was desirable to develop an independent synthesis for cAMPS.In this publication evidence is presented that the compound synthesized previously (Eckstein, 1970) was adenosine 5'-.Smethylphosphorothioate and that cAMPS can be obtained by reacting adenosine S'-O.O-bisf/z-nitrophenyOphosphorothioate with potassium zerz-butoxide. The interactions of cAMPS with 3',5'-cyclic nucleotide phosphodiesterases as well as with cAMP-dependent protein kinase from beef heart are described. Experimental SectionGeneral Procedures and MaterialsElectrophoresis was performed using Schleicher and Schiitl 2043 b (washed) paper in 0.1 M triethylammonium bicarbonate (pH 7.5) or borate buffer (pH 10) at 2000 V. The same paper was used for paper chromatography. Ultraviolet absorption measurements were carried out using a Zeiss PMQ II which was equipped with a Servogor recorder for kinetic stud-3From the Max-Planck-Instituí für experimentelle Medizin, Abteilung Chemie. Gottingen, Germany (F. F..