Esolation of 3-(2-carboxyethyl)thymine following in vitro reaction of beta-propiolactone with calf thymus DNA.

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from beta-propiolactone (BPL) and dThd 5'P at pH 9.0--9.5 via the intermediate 3-(2-carboxyethyl)-thymidine-5'-monophosphoric acid (3-CEdThd5'P). 3-CEdThd5'P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100 degrees C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3'-5')-thymidine gave 3-(2-carboxyethyl)thymidylyl-(3'-5')-3-(2-carboxyethyl)-thymidine (approximately 3%). Phosphotriester formation was not detected.