Enzymatic synthesis of the cAMP antagonist ( Rp)-adenosine 3′,5′-monophosphorothioate on a preparative scale

• Published in: Analytical biochemistry Vol. 188; no. 1; pp. 86 - 90
• Main Authors: Van Lookeren Campagne, Michiel M., Díaz, Fernando Villalba, Chason, Kevin W., Kessin, Richard H.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.07.1990; Elsevier
• Subjects: adenylate cyclase; Adenylyl Cyclases - isolation & purification; Adenylyl Cyclases - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Bordetella pertussis; Bordetella pertussis - enzymology; Bordetella pertussis - growth & development; cyclic AMP; Cyclic AMP - analogs & derivatives; Cyclic AMP - antagonists & inhibitors; Cyclic AMP - chemical synthesis; Cyclization; Drug Stability; Fundamental and applied biological sciences. Psychology; Kinetics; Nucleic acids; Nucleic bases, nucleotides; Substrate Specificity; Thionucleotides - chemical synthesis; Enzymatic synthesis; Adenylate cyclase; Preparative scale; Enzyme; Analog; Cyclic AMP; Nucleotide; Bacteria; Antagonist; Bordetella pertussis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(90)90531-D

( 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%.