Identification of unexpected modifications of fluorescein-labeled oligodeoxynucleotides by nuclease P1 digestion and mass spectrometric techniques

Fluorescein-labeled oligodeoxynucleotides (ODNs) from automated synthesis c ommonly produce multiple peaks in high performance liquid chromatography (H PLC) chromatograms, We found that these peaks are due to chemical modificat ions of the ODNs instead of the common perception of isomers, To identify t he modifications, a model ODN, fluorescein-T-25, was synthesized and five c ompounds were isolated. Nuclease P1 (NP1) digestion was employed to cleave these compounds into nucleotides and fluorescein-nucleotides in order that the modifications be determined by mass spectrometry (MS). Analyses of NP1 digestion products containing fluorescein by MS revealed the expected produ ct F1-T (M) and four unexpected compounds with MWs at M-1, M-17, M-16 and M + 16, respectively. Collision-induced dissociation (CID) spectra of these digestion products indicate that all modifications occur on the thiourea li nkage [-NH-C(=S)-NH-] to the fluorescein moiety and the adjacent phosphate group, and the modifications were determined. The modifications were also c onfirmed by accurate mass measurement with Fourier transform mass spectrome try (FT-MS), by the synthesis of a reference compound, and by a mechanistic study using model compounds. These results demonstrate the power of the ma ss spectrometric techniques by determining the structures of two pairs of O DNs with MW difference of 1 Da, The results also suggest that fluorescein p hosphoramidite with a thiourea linkage is not appropriate for the automated synthesis of fluorescein-labeled ODNs of high purity. Copyright (C) 2000 J ohn Wiley & Sons, Ltd.