Electrospray ionization collision induced dissociation of thieno[3,2-d]pyrimidine derivatives

2,4-substituted thieno[3,2-d]pyrimidines, with different l-amino acid methyl ester substituent on 4-C and methyl or methylthio substituent on 2-C, showed a complex and analytically valuable fragmentation behaviour under positive ion electrospray ionization collision-induced dissociation ((+)-ESI-CID) mass spectrometry. Protonated 2,4-substituted thieno[3,2-d]pyrimidines firstly lose a neutral fragment C2H4O2 (MS2) and the ion [M + H - 60]+ thus formed, then dissociates by losing of extended series of neutral molecule fragmentation from amino acid methyl ester substituent, in competition with the expulsion of CS or CH3SH and other direct bond scissions. Moreover, losing of NH3, CHN and CH2NH from the precursor ions of [M + H - 60]+ were proposed to occur via rearrangements. The formation of diagnostic product ions and the series neutral eliminations provided information about the properties of substituents, assisting their characterization via MS/MS, high-resolution mass spectrometry, the hydrogen/deuterium exchange experiment and density functional theory (DFT) calculations. The proposed fragmentation patterns of these compounds give an insight into the understanding of gas-phase reactions in mass spectrometric studies of thieno[3,2-d]pyrimidine derivatives.