Milligram‐Scale Enzymatic Synthesis of Base‐Modified DNA through Primer Extension: Polymerase Synthesis and NMR Structures of DNA Containing Phenyl‐Substituted Nucleobases

Milligram (≈200 nmol) scale enzymatic synthesis of base‐modified DNA is developed based on primer extension using easily accessible and cheap KOD (exo–) DNA polymerase with optimized protocol and streamlined isolation. The methodology is applied for synthesis of 31‐mer and 18‐mer DNA containing 5‐phenylpyrimidine or 7‐phenyl‐7‐deazapurine nucleobases using the corresponding phenyl‐modified dN Ph TP s in the PEX reactions. For the 18mers, the isolated yields are 24–61% of modified DNA samples of good purity, whereas for 31mers the samples contain minor amounts or shorter or longer impurities. The obtained amounts of modified DNA samples are sufficient for NMR structural analysis and CD spectroscopy that reveal only minor differences from B‐DNA structure of the same non‐modified DNA duplexes.