2',5'-Oligoadenylates chiral at phosphorus: enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioate trimer and tetramer analogues synthesized from (SP)-ATP alpha S.
The enzymatic synthesis and characterization of (RP)-2',5'-AMPS trimer and tetramer (SP)-5'-O-(1-thiotriphosphates) from chirally substituted (SP)-[alpha-35S]ATP alpha S by 2',5'-oligoadenylate synthetase from interferon-treated L cell extracts are described. The (RP)-ATP alpha S isomer is not a substrate for the synthetase. The identification of the trimer and tetramer analogues (molar ratio 70:30) was accomplished by high-performance liquid chromatography and subsequent separation by charge using DEAE-cellulose thin-layer chromatography. The digestion of the analogue by snake venom phosphodiesterase I (SVPD) to [alpha-35S]ATP alpha S and [35S]AMPS but not by T2 RNase demonstrated the presence of the 2',5' linkage. The assignment of RP configuration of the 2',5'-phosphorothiodiester linkage was based on the highly specific stereoselectivity of SVPD for RP diastereomers [Burgers, P. M. J., & Eckstein, F. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4978-4800; Bryant, F. R., & Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Nelson, P. S., Bach, C. T., & Verheyden, J. P. H. (1984) J. Org. Chem. 49, 2314-2317]. This suggests that the synthesis of the phosphorothioate analogues proceeded via inversion of configuration at the chiral phosphorus of (SP)-ATP alpha S. The putative (RP)-2',5'-AMPS tetramer (SP)-5'-O-(1-thiotriphosphate) displaced the 2',5'-p3A4[32P]pCp analogue from 2',5'-oligoadenylate-dependent endonuclease 5 times more efficiently than did equimolar concentrations of authentic 2',5'-adenylate tetramer triphosphate. Furthermore, in studies using the calcium phosphate coprecipitation technique, the 2',5'-phosphorothioate trimer and tetramer analogues inhibited protein synthesis better than did 2',5'-adenylate trimer and tetramer triphosphates.(ABSTRACT TRUNCATED AT 250 WORDS)
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism; D000227:Adenine Nucleotides / Q000096:biosynthesis*; D000818:Animals; D004355:Drug Stability; D007370:Interferon Type I / Q000494:pharmacology; D007700:Kinetics; D007739:L Cells / Q000201:enzymology; D051379:Mice; D009841:Oligonucleotides / Q000096:biosynthesis*; D009843:Oligoribonucleotides / Q000096:biosynthesis*; D010761:Phosphorus Radioisotopes; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013873:Thionucleotides / Q000096:biosynthesis*
D000227:Adenine Nucleotides; D007370:Interferon Type I; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; C023505:2',5'-oligoadenylate; D015088:2',5'-Oligoadenylate Synthetase