The α-thio and/or β-γ-hypophosphate analogs of ATP as cofactors of T4 DNA ligase

•The pure diastereomers of ATPαS, β,γ-hypo-ATPαS and β,γ-hypo-ATP (oxo) were tested as cofactors for the T4 DNA ligase.•Only ATPαS-SP and β,γ-hypo-ATP (oxo) were found to be efficient DNA end-joining, however none of them has an inhibitor’s activity.•Newly synthesized β,γ-hypo-ATP (oxo) express lower affinity towards tested T4 DNA ligase, when compared to ATP.

T4 DNA ligase is one of the most commonly used enzymes for in vitro molecular research and a useful model for testing the ligation mechanism of ATP-dependent DNA ligation. To better understand the influence of phosphate group modifications in the ligation process, a series of ATP analogs were tested as cofactors. P-diastereomers of newly developed β,γ-hypo-ATPαS (thio) and β,γ-hypo-ATP (oxo) were synthesized and their activity was compared to ATPαS and their natural precursors. The evaluation of presented ATP analogs revealed the importance of the α-phosphate stereogenic center in ATPαS for the T4 DNA ligase activity and sheds new light on the interaction between ATP-dependent DNA ligases and cofactors.

Graphical abstractThe T4 DNA ligase accepts ATPαS-SP and β,γ-hypo-ATP (oxo) for DNA end-joining reaction, but does not accept ATPαS-RP neither both epimers of β,γ-hypo-ATPαS as cofactors.Figure optionsDownload full-size imageDownload as PowerPoint slide