HPLC analysis of reaction products
Reaction mixtures contained 2 mM CEMS and 2 mM purinethiol. At completion of reaction, samples were diluted five-fold with 5.0 mM Tris, pH 8.0, and analyzed by HPLC. A. The reaction of CEMS with 6MP was analyzed with the absorbance detector set at 293 nm, selective for the expected adduct. Trace 1 is a representative chromatogram from the complete reaction mixture; trace 2, reaction mixture lacking CEMS; trace 3, reaction mixture lacking purinethiol; traces are offset by arbitrary amounts along the y-axis for clarity. B. The adduct eluting at 15.0 min was collected from multiple runs and analyzed by mass spectrometry as described in Materials and Methods. C. The reaction of CEMS with DTP was analyzed with the absorbance detector set at 320 nm, selective for the expected adduct. Traces labeled as in panel A. D. The adduct eluting at 4.4 min was collected form multiple runs and analyzed by mass spectrometry.