Abstract The ability of acrylonitrile to cyanoethylate 13 nucleosides was examined. Only pseudouridine, inosine, and 4-thiouridine were found to react rapidly, yielding N1-cyanoethyl pseudouridine, N1-cyanoethyl inosine, and S-cyanoethyl 4-thiouridine, respectively. At much slower rates, a reaction with position N3 of uridine, ribothymidine, and N1-cyanoethyl pseudouridine was observed, and at very slow rates a 2'- (or 3'-) cyanoethyl derivative was produced with adenosine, guanosine, cytidine, and N1-cyanoethyl inosine. The variation of rate constant with pH showed that the rapidly reacting nucleosides must be in the anionic form for reaction to occur. The spectral, chromatographic, and electrophoretic properties of the derivatives were determined and used to assign the structures as given. The stability of the cyanoethyl derivatives to alkali and acid was examined and found to be sufficient to allow the application of standard procedures for the detailed nucleotide analysis of cyanoethylated transfer RNA.
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