Synthesis, Photophysical Properties, and Enzymatic Incorporation of an Emissive Thymidine Analogue

Herein, we describe the synthesis, photophysical characterization, and DNA incorporation of a fluorescent nucleobase analogue, thieno[3,4-d]-pyrimidine T-mimic deoxynucleoside, thdT. thdT exhibits comparable thermal stability and selectivity of base pairing to that of natural T and noteworthy photophysical behaviors depending on its microenvironments in DNA. A thio-analogue of thymidine triphosphate, thdTTP, has been synthesized and enzymatically incorporated into DNA using naturally occurring polymerases and replication systems with assistance from dTTP. The present results show the utility of thdT as a fluorescent surrogate of thymidine for further applications.