A hydrophobic unnatural nucleoside of 4-propynylpyrrole-2-carbaldehyde (designated as Pa') was synthesized to improve its affinity with a pairing partner, 9-methyl-imidazo[(4,5)-b]pyridine (Q), in enzymatic incorporation. In single-nucleotide insertion experiments using the Klenow fragment, the substrate of Pa' (dPa'TP) was efficiently incorporated opposite Q in the template strand, as compared to the incorporation of pyrrole-2-carbaldehyde (dPaTP), which was previously developed.