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 α-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)-ATPαS). The adenylate cyclase from Bordetella pertussis, partially purified by calmodulin affinity chromatography, cyclizes ( Sp)-ATPαS approximately 40-fold more slowly than ATP, but binds ( Sp)-ATPαS 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%.