Synthesis of Cy5-Labelled C5-Alkynyl-modified cytidine triphosphates via Sonogashira coupling for DNA labelling

•Novel water-soluble cyanine dyes with terminal alkyne group were synthesized.•Highly efficient approach of the single-step Sonogashira-type cross-coupling reaction for the synthesis of 2′-deoxycytidine-5′-triphosphates bearing a novel cyanine dye attached via an acetylene linker using freeze–pumpthaw degassing methodology was developed.•Dye-modified 2′-deoxycytidine-5′-triphosphates containing an alkynyl linker with a carboxamide fragment is the most efficient substrate for Taq and Vent (exo-) DNA polymerases.