title: Enzymatic synthesis of the cAMP antagonist (Rp)-adenosine 3',5'-monophosphorothioate on a preparative scale.
abstract: (Rp)-Adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) is a highly specific antagonist of the cAMP-dependent protein kinase from eukaryotic cells and is a very poor substrate for phosphodiesterases. It is therefore a useful tool for investigating the role of cAMP as a second messenger in a variety of biological systems. Taking advantage of stereospecific inversion of configuration around the alpha-phosphate during the adenylate cyclase reaction, we have developed a method for the preparative enzymatic synthesis of the Rp diastereomer of adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) from the Sp diastereomer of adenosine 5'-O-(1-thiotriphosphate) ((Sp)-ATP alpha S). The adenylate cyclase from Bordetella pertussis, partially purified by calmodulin affinity chromatography, cyclizes (Sp)-ATP alpha S approximately 40-fold more slowly than ATP, but binds (Sp)-ATP alpha S with about 10-fold higher affinity than ATP. The triethylammonium salt of the reaction product can be purified by elution from a gravity flow reversed-phase C18 column with a linear gradient of increasing concentrations of methanol. Yields of the pure (Rp)-cAMPS product of a synthesis with 2 mg of substrate are about 75%.
authors.affiliation: Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
authors.forename: M M
authors.initials: MM
authors.lastname: Van Lookeren Campagne
authors.forename: F
authors.initials: F
authors.lastname: Villalba Díaz
authors.forename: K W
authors.initials: KW
authors.lastname: Chason
authors.forename: R H
authors.initials: RH
authors.lastname: Kessin
mesh_terms: D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism; D001886:Bordetella pertussis / Q000201:enzymology / Q000254:growth & development; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis; D003500:Cyclization; D004355:Drug Stability; D007700:Kinetics; D013379:Substrate Specificity; D013873:Thionucleotides / Q000138:chemical synthesis*
chemical_list: D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases