Fluorescent 2‐aza‐ε‐adenosine derivatives of polyadenylic acid, polyuridylic acid, and polyinosinic acid

Abstract

SynopsisA new fluorescent analog of adenosine, 1f16-etheno-2-aza-adenosine, has been incorporated into polynucleotides by polynucleotide phosphorylase polymerization of 1,N6-etheno-2-aza-adenosine-5'-diphosphate and adenosine-5'-diphosphate, uridine-5'-diphosphate, or inosine-5'-diphosphate. These new oligonucleotides possess high fluorescence when excited a t 358 nm and emit at 495 nm. The ratio of the fluorescent and nonfluorescent portions of the copolymer can be controlled by the initial composition of the 2-aza-6-adenosine-diphosphate and the corresponding nucleoside diphosphate. Fluorescent copolymers with a ratio varying from 1.6 to 35 have thus been synthesized. The physicochemical study of copolymers containing less than 10% of the 1,N6-etheno-2-aza-adenosine moiety showed that they are similar to poly(A), poly(U), or poly(1). Therefore, fluorescence and polarization study of the 1,N6-etheno-2-aza-adenosine residues that have been incorporated into the copolymer provides a sensitive indicator for the structure of the copolymer. Potentially these new copolymers may provide unique roles in probing the structure of poly(C) and poly(A) in cellular mRNA. INTRODUCTIONFluorescent labeling of macromolecules has been useful in providing a better understanding of the chemical and physical interactions of biopolymers. Perhaps the best known example is the conjugation of a fluorescent molecule to a pr0tein.l The fluorescent labeling of nucleic acids should be equally useful for conformation studies, as well as for studies of nucleic acid-nucleic acid or nucleic acid-protein interactions.Recently the syntheses of a series of fluorescent adenosine analogs based on 1,N6-etheno-2-aza-adenosine (aza-c-Ado)2 and its n u c l e o t i d e~~,~ have been accomplished in our laboratory. These novel nucleotides have been found to mimic closely in enzymatic reaction the natural adenosine analogs. In addition, their emission maximum at 495 nm (when excited a t 358 nm) fits the requirement of most fluorescent microscopes, and they are therefore suitable for cytochemical research. In the course of this work, we found that the 1,N6-etheno-2-aza-adenosine 5'-diphosphate (Scheme I) could Biopolymers, Vol. 18, 1389Vol. 18, -1405Vol. 18, (1979 be readily polymerized in the presence of E. coli polynucleotide phosphorylase (PNPase) to a fluorescent poly(2-aza-e-adenylic acid).4Extension of this finding to a histochemical method for the demonstration of a similar reaction in mammalian tissue has been proposed, and preliminary observation of this activity in both normal and cancerous human tissues has been r e p~r t e d .~ The work has also been extended to the synthesis of copolymers of this new fluorescent nucleotide with adenylic acid, uridylic acid, or inosinic acid. The present paper reports both the synthesis and physicochemical and spectroscopic studies of these new fluorescent copolymers. EXPERIMENTALThe synthesis of 2-aza-c-adenosine-5'-diphosphate (2-aza-e-ADP) was reported previously.2 Polyadenylic acid, polyuridylic acid, pol...