Next, we focused on hydroxylation, nitration, and amination reactions with 1, 14, and 15. Hydroxylation of 14 was accomplished in two steps. First, treatment of 14 with hydrogen peroxide (30%) in sodium carbonate generated 2,3-epoxidemenadione (21i), which was subsequently treated with sulfuric acid to yield hydroxy-menadione (21) in an overall yield of 62%. Several attempts were made to synthesize methoxy-menadione (13) from 21 using methyl iodide and silver(I) oxide, however it yielded a mixture of inseparable products -13 and 4methoxynaphthalene-1,2-dione. Therefore, 13 was obtained from a commercial source and no other attempts were made to derivatize the hydroxyl in 21. Introduction of the nitro-functionality into 14 involved treatment with a mixture of concentrated nitric acid and sulfuric acid. The orange crude product was recrystallized in ethanol to yield nitro-menadione (22) in low yields (24%). Uncrystallized product was observed in the filtrate but was discarded rather than conducting a large-scale biphasic extraction involving concentrated nitric acid. Pure 22 was subsequently reduced with 10% Pd/C and H2 (g) in quantitative yields to produce amine-menatione (23). In addition, ACNQ (5) was obtained through treating 1 with ammonium chloride following previously reported methods. Following literature precedent, primary (e.g., methyl and ethyl amine) and secondary (e.g., piperidine and morpholine) amines were added to 14 through a perchloric acid catalyzed reaction, yielding mediators 24 -27 in moderate yields (20 -44%). Lastly, similar conditions on 15 yielded methylamine-naphthoquinone (28) and morpholinenaphthoquinone (29) in moderate yields (38 -57%). Many chemical properties of the mediators are easily obtained using available software or online tools, such as ChemDraw and MolGpka, which were used to calculate LogD, and to determine tPSA (Table and). However, calculating the E o and Ndh2 binding affinity (Ki) of a compound is considerably more challenging. For E o , previous literature established that there is a linear relationship between the energy difference of the ground state quinone and radical anion, and experimentally determined one electron (1e -) E°. Therefore, we used Density Functional Theory (DFT) to calculate this energy on all mediators (Table ) and the E o was experimentally measured for a diverse subset (twenty mediators) of the library (Fig. ). Graphing the theoretical molecular energy difference versus E o yielded a line of best fit (R 2 = 0.85) that was used to calculate the E o for the remaining mediators (Fig. ). All E o were determined to fall between -1.44-(-0.16) V (Table ). This method allowed us to calculate the E o of 1, which posed a challenge experimentally due to the difficulty to obtain the molecule commercially in the quinone form. Finally, we used molecular docking tools, specifically AutoDock Vina, to approximate each mediator's Ndh2 binding affinity. Although this predicted free energy of binding (ΔGcomp) is not the same as binding affinity, it correlates with the mediator's ability to bind to Ndh2. Experimental determination of Ki values is not trivial as Ndh2 is a membrane protein, thus significantly complicating protein expression. Ndh2-dependent EET activity in L. plantarum was quantified using an established iron(III) oxide nanoparticle reduction assay, which uses ferrozine to quantify the amount of Fe 3+ that is converted to Fe 2+ by L. plantarum (Fig. ). Absent of any exogenous mediator, no reduction of Fe 3+ to Fe 2+ is observed. Although L. plantarum is incapable of producing quinones, the bacterium does have the ability to metabolize these compounds. Specifically, DmkA has the ability to prenylate unoccupied 2-or 3positions of naphthoquinone analogs to produce demethylmenaquinone analogs. To ensure the observed EET is mediated by Ndh2, we compared the EET activity between two previously developed mutant strains of L. plantarum (L. plantarum ∆dmkA∆ndh1 and L. plantarum ∆dmkA∆ndh1/2). Removal of the genes dmkA and ndh1 eliminates EET side-reactions that may modify, or consume the mediator, ensuring EET depends primarily on the interaction between Ndh2 and the given mediator. All mediators were evaluated for EET activity at 5 μM, counter screened for toxicity at 5 and 50 μM, and evaluated for chelation properties (Fig. ). As additional confirmation that observed EET activity is Ndh2-dependent, we conducted additional iron(III) oxide nanoparticle assays with all quinones and two additional strains of L. plantarum ∆dmkA∆ndh1/2: L. plantarum expressing a functional Ndh2 (∆dmkA∆ndh1/2 + pSL93) and with a point mutant (∆dmkA∆ndh1/2 + pRA01). This point mutation reduces activity in the homologous Ndh2 of C. thermarum, strongly suggesting that the activity in an analogous mutation in L. plantarum Ndh2 will alter EET activity. Overall, just about half of the quinone library was able to perform Ndh2-dependent EET (Fig. ). Both naphthoquinones and benzoquinones were capable of mediating EET, while none of the anthraquinones were selective for Ndh2-dependent EET. These patterns of Ndh2-dependent EET were also observed in the assay comparing the point mutant Ndh2 to functional Ndh2 (Fig. ). In addition, abiotic controls of the iron(III) oxide nanoparticle assays showed no significant background activity (Fig. ). The top ten performing mediators in Fig. included seven naphthoquinones (3, 5, 7, 8, 19, 21, and 23) and three benzoquinones (36, 37, and 39). All seven of the naphthoquinones and two of the benzoquinones contain an electron-donating group (hydroxyl, halogen, methoxy, or amine) that is conjugated to the quinone moiety. In addition, nine of the top ten mediators possess a hydrogen bond donor (e.g., methoxy, hydroxyl, cyano, carboxylic acid, amine) in either the 2-or 3- As predicted by pKa calculations; All E o listed were calculated using the linear relationship between the energy difference of the ground state quinone and radical anion compared to select experimentally determined one electron E o using an Ag/Ag + reference electrode; Predicted free energy of binding which was calculated using pharmacophore modeling; position. Closer analysis of the molecular docking poses for EET active compounds with hydrogen bond donating substituents (3, 21, 23) revealed the hydrogen bond donor substituent is positioned within 2.7 -3.0 Å of the sidechain amide carbonyl on asparagine-387 (Fig. ). Conversely, for alkylated analogs (13, 24, 26), the lowest energy poses have the mediator rotated and not within hydrogen bonding distance of asparagine-387 (Fig. ). Therefore, indicating a hydrogen bond donor on the mediator might promote favorable interactions within the Ndh2 active site and could be stabilizing the electron transfer.