An unnatural base pair that is replicated and transcribed with good efficiency would lay the foundation for the long term goal of creating a semisynthetic organism, but also would have immediate in vitro applications, such as the enzymatic synthesis of site-specifically modified DNA and/or RNA. One of the most promising of the unnatural base pairs that we have identified is formed between d5SICS and dMMO2. The ortho substituents of these nucleotides are included to facilitate unnatural base pair extension, presumably by forming a hydrogen-bond with the polymerase, but the synthesis of the unnatural base pair still requires optimization. Recently, we have shown that meta and/or para substituents within the dMMO2 scaffold can facilitate unnatural base pair synthesis, although the mechanism remains unclear. To explore this issue, we synthesized and evaluated several dMMO2 derivatives with meta-chlorine, -bromine, -iodine, -methyl, or -propinyl substituents. Complete characterization of unnatural base pair and mispair synthesis and extension reveal that the modifications have large effects only on the efficiency of unnatural base pair synthesis and that the effects likely result from a combination of changes in steric interactions, polarity, and polarizability. The results also suggest that functionalized versions of the propinyl moiety of d5PrM should serve as suitable linkers to site-specifically incorporate other chemical functionalities into DNA. Similar modifications of d5SICS should allow labeling of DNA with two different functionalities, and the previously demonstrated efficient transcription of the unnatural base pair suggests that derivatives might similarly enable site-specific labeling of RNA.