Because the motivation behind our development of PGD was to build libraries for screening campaigns, we sought assays that could be used to evaluate the new compounds. Protein translation was a logical starting point in a search for potential activity because the desosamine moiety in the initial PGD library is present on antibacterial translation inhibitory macrolides such as erythromycin, clarithromycin, azithromycin, etc. It was likely our compounds would have some affinity to the negatively charged rRNA under physiological pH which would render the amine protonated. We considered it unlikely that the small library would recapitulate the activity of those macrocycles. Rather, we hoped to find compounds that might inhibit protein translation, but were structurally simpler than erythromycin itself. Importantly, compounds with these qualities would be amenable to development in terms of medchem to increase potency and tune physicochemical properties. In vitro transcription-translation assays have been established primarily for the small-scale synthesis of new proteins. The assay also has been used to identify inhibitors of this process. There is significant potential for new compounds that can stop, or stall, protein translation. Stalling has been the primary mechanism of action of the macrolides as antibacterials. Recently, a selective, small molecule moldulator of the human ribosome as it translates PCSK9 was identified. This exciting result opens the possibility of identifying other modulators of human translation. PGD compound libraries, like the one reported here, could be candidates for such studies. Experimentally, the PURExpress cell-free transcription/translation system (New England Biolabs, Inc) was adapted so that fluorescence would provide a quantitative estimate of protein synthesis based on the signal produced from maturing emerald green fluorescent protein, EmGFP. characteristic fluorescence emission was observed in the absence of vector, indicating that the signal in the assay came primarily from EmGFP rather than being intrinsic to the PURExpress kit (Fig. ). Although there were slight variations between PURExpress lots used in the study, addition of 175-400 ng of vector DNA to the reaction mixture consistently yielded a robust signal. Addition of vector to the assay initiated the biosynthesis of EmGFP and produced a continuous increase in fluorescence intensity at 507 nm that reached a plateaued after three or four hours (Fig. ). Library compounds 7 and 10-12 were then tested for their ability to inhibit EmGFP production at a single concentration (25 μg/mL in DMSO), where total DMSO concentration was <1% by volume.