2‐Allyl‐ and Propargylamino‐dATPs for Site‐Specific Enzymatic Introduction of a Single Modification in the Minor Groove of DNA

A series of 2‐alkylamino‐2′‐deoxyadenosine triphosphates (dATP) was prepared and found to be substrates for the Therminator DNA polymerase, which incorporated only one modified nucleotide into the primer. Using a template encoding for two consecutive adenines, conditions were found for incorporation of either one or two modified nucleotides. In all cases, addition of a mixture of natural dNTPs led to primer extension resulting in site‐specific single modification of DNA in the minor groove. The allylamino‐substituted DNA was used for the thiol‐ene addition, whereas the propargylamino‐DNA for the CuAAC click reaction was used to label the DNA with a fluorescent dye in the minor groove. The approach was used to construct FRET probes for detection of oligonucleotides.