Mechanochemistry offers a green, solid-state pathway for designing nanomaterials, while chemical aging provides precise control under mild conditions. This study combines both methods to create nonstoichiometric bismuth oxide nanosheets with exceptional adsorption and photocatalytic performance toward persistent organic pollutants.Detailed structural and compositional analyses revealed that strain-induced defect formation during grinding followed by chemical delamination under aging leads to the formation of defect-rich Bi₂O₂.₃₃ nanosheets. This mechanochemical-aging synthesis provides new insight into two-dimensional metal oxide design and expands the potential for sustainable nanomaterial production.<blockquote><h4>The original article</h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsmaterialsau.5c00104">Mechanochemical-Aging Synthesis of Bismuth Oxide Nanosheets for Photocatalysis</a></h4>Delaney J. Hennes, Luke T. Coward, Chase G. Thurman, Oksana Love*, and Pin Lyu*ACS Mater. Au 2025, XXXX, XXX, XXX-XXX<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsmaterialsau.5c00104">https://doi.org/10.1021/acsmaterialsau.5c00104</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Mechanochemistry, old techniques with new perspectives, has been reawakening the passion for exploring solid-state approaches toward chemical transformations and materials synthesis in a green and sustainable manner, due to its intrinsic solvent-free and eco-friendly nature. <a href="javascript:void(0);">(1−8)</a> More importantly, the driving force of the mechanochemical process, mechanical force, has provided a fundamentally different reaction pathway by promoting intensive, well-oriented, and effective molecular collisions and creating molecular strains and structural defects, <a href="javascript:void(0);">(6,9−13)</a> compared to the conventional thermal or electrostatic driving forces. This perspective has opened new avenues for well-defined nanomaterial synthesis with unique structure–property relationships that the conventional solution-based methods cannot achieve, as well as in a greener way. <a href="javascript:void(0);">(4,14−17)</a> Recent reports have demonstrated great potential in designing metal and metal oxide nanoparticles, <a href="javascript:void(0);">(17,18)</a> nanocomposites, <a href="javascript:void(0);">(19)</a> and hybrid porous materials such as metal–organic frameworks (MOFs) and covalent organic frameworks (COFs) with desired properties. <a href="javascript:void(0);">(20−22)</a>In terms of 2D nanostructure design, two traditional approaches─bottom-up and top-down synthesis─have been widely developed in various synthesis strategies, offering precise control over size, shape, morphology, and dimensions. <a href="javascript:void(0);">(23,24)</a> The formation mechanism behind the scenes can be monitored and reasonably interpreted by advanced in situ spectroscopic <a href="javascript:void(0);">(25−27)</a> or microscopy methods, <a href="javascript:void(0);">(28−30)</a> to identify the critical factors in the structure transformation that lead to property-on-demand synthesis. Specifically, in 2D metal oxide nanosheet synthesis (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsmaterialsau.5c00104#sch1">Scheme 1</a>), chemical vapor deposition, solvothermal method, and chemical exfoliation represent three typical methods. The first two methods start with molecular or atomic precursors for self-assembly growth, while the last method utilizes bulk layered host materials for layer-by-layer delamination. However, these methods rely on sophisticated instrumentation, harsh synthesis conditions, excess solvents, or toxic exfoliating chemicals, which limit their potential scalability.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Mater_Au_2025_Scheme_1_Comparison_of_Conventional_and_Mechanochemical_Aging_Methods_for_Bismuth_Oxide_Nanosheet_Synthesis_536967aa25.jpg" alt="ACS Mater. Au 2025: Scheme 1. Comparison of Conventional and Mechanochemical-Aging Methods for Bismuth Oxide Nanosheet Synthesis" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Mater_Au_2025_Scheme_1_Comparison_of_Conventional_and_Mechanochemical_Aging_Methods_for_Bismuth_Oxide_Nanosheet_Synthesis_536967aa25.jpg 245w," sizes="100vw" width="1000" height="487">Mechanochemical synthesis, with its all-solid-state, solvent-free nature, provides an alternative approach. This approach introduces an external applied physical force to initiate intense molecular interactions for mixing and growing (bottom-up) or to spread the shear stress or shock impact uniformly for deformation or delamination (top-down). It has been elegantly demonstrated in various traditional two-dimensional materials syntheses, such as graphene, <a href="javascript:void(0);">(31)</a> MXenes, <a href="javascript:void(0);">(32)</a> and transition-metal dichalcogenides. <a href="javascript:void(0);">(33)</a> Specifically, for bismuth-based materials, nanosheets of bismuthene, <a href="javascript:void(0);">(34)</a> bismuth oxyhalides, <a href="javascript:void(0);">(35)</a> and bismuth nitrates <a href="javascript:void(0);">(36)</a> have been reported previously with the mechanochemical methods. Meanwhile, very few reports are focused on metal oxide 2D nanostructures, probably due to the relatively higher interlayer interactions in those host materials. Thus, a follow-up process is needed to accelerate delamination of the mechanochemically produced bulk materials into the desired 2D nanosheet structure. Recently, Moores’s and Friščić’s groups have successfully demonstrated the combination of mechanochemistry and aging for the extraction and functionalization of biomass derivatives, <a href="javascript:void(0);">(37,38)</a> and functionalized inorganic metal or binary nanoparticle synthesis. <a href="javascript:void(0);">(39,40)</a> Aging, as a spontaneous diffusion-controlled process, can be controlled by the chemical reactivity and solution environment to induce material transformation, further advancing to the targeted structures while minimizing reagent and energy use. <a href="javascript:void(0);">(40)</a>In this work, we developed a mechanochemical-aging process to synthesize nonstoichiometric bismuth oxide (Bi2O2.33) nanosheets, which demonstrated excellent performance in the adsorption and photodegradation of perfluorooctanoic acid (PFOA). Through the observation of morphological, crystal structure, and surface chemistry changes during the aging process, we propose a strain-to-defect growing mechanism. Mechanical force exerted on metal precursors and reducing agents during solid-state grinding can induce rapid and intense molecular collisions, leading to the generation of strain-rich metallic nanostructures. The following aging process, occurring in an aqueous surfactant-deficient environment, can delaminate the bulk structure into well-defined 2D nanosheets over time with the assistance of ambient oxygen. This approach could potentially be applied to other 2D metal oxide nanostructures.<h4>Experimental Methods</h4><h5>Chemicals and Characterizations</h5>All chemicals and reagents were used without any purification. Bismuth(III) nitrate pentahydrate (99.999%, trace metal basis), PVP (M.W. 40,000), and sodium borohydride (99%, powder) were purchased from Thermo Scientific Chemicals. The morphology and elemental mapping analysis of bismuth oxide nanosheets (Bi2O2.33 nanosheets) was examined by scanning electron microscopy (SEM, JSM-IT700HR, 15 kV, JEOL) with energy dispersive X-ray spectroscopy (EDS, 0–20 kV energy window, Ultim Max 40: UVA13205 detector, Oxford Instruments). The evolution of the local crystalline structure of Bi2O2.33 nanosheets was monitored by a powder X-ray diffraction pattern (PXRD, Bruker D2 PHASER Benchtop XRD, Cu tubes, 30 kV, 10 mA) and was processed with DIFFRAC.EVA data analysis software with the Crystallography Open Database (rev.278581). The surface status of functional groups was determined by Fourier transform infrared spectroscopy (FTIR, Shimadzu <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=1&amp;search=IRSpirit">IRSpirit</a>, 16 scans collected with a resolution of 4 cm–1) with the attenuated total reflectance (ATR) mode. The quantification of the PFOA (C7F15–COOH) and its degradation intermediates, perfluoroheptanoic acid (PFHpA, C6F13–COOH) and perfluorohexanoic acid (PFHxA, C5F11–COOH), was determined by liquid chromatography–mass spectrometry (LC-MS, <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=3&amp;search=LCMS-8040">LCMS-8040 Triple Quad LC-MS/MS</a>, Shimadzu Scientific Instruments) in negative electrospray ionization mode. Separation of analytes occurred with the use of a binary solvent gradient between 5 mM ammonium acetate in H2O and methanol that ramped from 5% methanol at 0–3 min to 40% methanol from 3 to 16 min and 80% methanol after 16 min. Each liquid was passed through a Phenomenex C18 column (1.8 μm, 100 × 2.1 mm) at a constant oven temperature of 40 °C with a solvent flow rate of 0.25 mL per min. The elution of PFOA occurred at 15.7 min, PFHpA at 13.2 min, and PFHxA at 11.6 min. A 13C isotope-labeled PFOA internal standard (Wellington Laboratories) was paired with a 12C native PFOA solution to quantify each sample’s concentration. The standard curve for PFOA quantification ranges from 0.5, 1, 5, 10, 25, 50, and 100 ppb, with a limit of detection of 3.6 ppb and a limit of quantification of 11.1 ppb.<h4>Results and Discussion</h4>To pinpoint the growing mechanism behind the mechanochemical-aging process, morphological, crystal structure, and surface chemistry changes are monitored by using SEM, XRD, and FTIR, respectively. First, after freshly grinding, the gray powder was sent for XRD testing, as shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsmaterialsau.5c00104#fig2">Figure 2</a>a as Sample (Day 1). All of the observed diffraction peaks match well with the standard PDF card of metallic bismuth, indicating the successful reduction of molecular bismuth precursors. There is a small peak around 12 degrees, which matches one of the broad peaks of PVP powder (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsmaterialsau.5c00104/suppl_file/mg5c00104_si_001.pdf">Figure S1</a>), which is anticipated at this stage since the PVP used in the precursor mixture is 5 times the weight of the bismuth salts and has not been washed away. There is another small peak around 29° that matches nonstoichiometric bismuth oxide, which will be discussed below. According to the previous report, <a href="javascript:void(0);">(50)</a> the overall hexagonal crystal structure with a rhombohedral unit cell (as shown by the gray connections in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsmaterialsau.5c00104#fig2">Figure 2</a>b) is commonly observed at ambient conditions for metallic bismuth. Two bilayers within the hexagonal structure are separated by relatively weak van der Waals-like interactions, which provide space and relatively low energy barriers for the following aging process. As follows, during the aging process, with the assistance of PVP surfactant solutions and dissolved ambient oxygen, the metallic Bi gradually oxidizes, and the crystal structure becomes more amorphous, as observed with 3 broad peaks of XRD patterns in Day 4’s and Day 7’s samples. The PVP peaks are not observed in these samples in the XRD, possibly due to the disruptions of the ordered structure in the powder form when they are dispersed in a low concentration solution (0.5 mM). Eventually, on Day 10, a fully oxidized sample is formed, with a crystal structure matching the nonstoichiometric Bi2O2.33 standard card, and no distinct peaks are observed for metallic Bi anymore. Additionally, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsmaterialsau.5c00104/suppl_file/mg5c00104_si_001.pdf">Figure S2</a> compares standard patterns for bismuth subcarbonate and three distinct phases of stoichiometric bismuth oxide with those of the Day 10 sample. None of these reference patterns match the observed pattern of the Day 10 sample, ruling out the presence of these phase impurities in our material. This mixed-valent (Bi2+ and Bi3+) electron-rich structure of nonstoichiometric Bi2O2.33 was previously reported in research using traditional synthesis methods mentioned above, which demonstrated excellent properties, such as UV-emitting photoluminescence, supercapacitance, ferromagnetism, and photocatalysis. <a href="javascript:void(0);">(41,43,47)</a> To the best of our knowledge, there have been no reports on the mechanochemistry-driven synthesis of this unique structure. To be specific, as shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsmaterialsau.5c00104#fig2">Figure 2</a>c, the overall tetragonal structure consists of a mixture of Bi2O3 and Bi2O2 layers within the lattice, possibly coming from the oxidation of metallic bismuth in the original bilayer structures before aging. This unique structure, featuring the stereochemically active lone pair of Bi3+ and the electrostatically electron-rich site of Bi2+, could potentially contribute to interlayer flexibility and defect formation, ultimately leading to the final nanosheet structure. This has been observed in other bismuth-based materials with preferred layered structures as well. <a href="javascript:void(0);">(51−53)</a><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Mater_Au_2025_Figure_2_Structural_evolution_for_the_mechanochemical_aging_synthesis_process_a6c739235e.jpg" alt="ACS Mater. Au 2025: Figure 2. Structural evolution for the mechanochemical-aging synthesis process. (a) XRD spectra of samples collected on Day 1 (freshly made) and Days 4, 7, and 10 with the standard PDF reference cards for metallic Bi and nonstoichiometric Bi2O2.33. Schematic presentation of the crystal structures of (b) metallic Bi and (c) nonstoichiometric Bi2O2.33." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Mater_Au_2025_Figure_2_Structural_evolution_for_the_mechanochemical_aging_synthesis_process_a6c739235e.jpg 245w," sizes="100vw" width="2100" height="1168">Moving forward, the SEM images of freshly made (Day 1) samples and samples at different intervals during the aging process were collected for tracking morphological changes, along with EDS mapping to confirm the elemental composition and compare the spatial distribution of these elements. As shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsmaterialsau.5c00104#fig3">Figure 3</a>, the bulk rod microstructure of metallic Bi after grinding and washing on Day 1 was observed with a relatively small amount of well-defined hexagonal thin nanosheets. These nanosheets have a relatively smooth surface, and the edge length is about 1 μm with a lateral width of 2 μm. The EDS full-range scan results also confirmed the dominant element compositions of carbon, oxygen, and bismuth in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsmaterialsau.5c00104/suppl_file/mg5c00104_si_001.pdf">Figure S3</a>, in which the carbon comes from both the carbon tape used in the sample preparation and the PVP polymer residues. For the EDS mapping in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsmaterialsau.5c00104/suppl_file/mg5c00104_si_001.pdf">Figure S4</a>, the distributions of Bi and O are relatively and consistently uniform across the sample, while the oxygen amount is significantly higher than the bismuth (estimated based on the spectra intensity counts and relative atomic mass). It is possible due to the residual PVP polymer still adsorbed on the surface, and is ready to participate in the next delamination process. This is also observed in the FTIR spectrum of O–H bending, C–H bending, and C–N stretching, possibly from the PVP, as shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsmaterialsau.5c00104/suppl_file/mg5c00104_si_001.pdf">Figure S5</a>, compared to the FTIR spectrum of PVP powder. It is noteworthy that very weak Bi–O bonding and a hydrophobic surface with almost no O–H stretching are also observed in Day 1’s sample, combined with the evidence of PVP, dominant metallic Bi peaks, and a small peak of Bi2O2.33 from the XRD analysis above. It is suggested that after freshly grinding, the metallic Bi bulk microrods formed as the aging precursor for the subsequent delamination process, and small amounts of oxide nanosheet structure began to form adjacent to the bulk rod, indicating a delamination pathway rather than nucleation from the aging solution.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Mater_Au_2025_Figure_3_Morphology_evolution_for_the_mechanochemical_aging_synthesis_process_9a69a3b684.jpg" alt="ACS Mater. Au 2025: Figure 3. Morphology evolution for the mechanochemical-aging synthesis process. SEM images of samples collected on (a–d) Day 1 (freshly made), (e–h) Day 4, (i–l) Day 7, and (m–p) Day 10." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Mater_Au_2025_Figure_3_Morphology_evolution_for_the_mechanochemical_aging_synthesis_process_9a69a3b684.jpg 205w," sizes="100vw" width="3000" height="2285"><h4>Conclusions</h4>In summary, we developed a mechanochemical-aging approach for the synthesis of nonstoichiometric Bi2O2.33 nanosheets, exhibiting excellent adsorption capacity and photocatalytic degradation performance toward forever chemicals. Different stages of morphological, crystal structure, and surface composition changes were observed and correlated to our proposed growth mechanism. Our mechanistic interpretation focuses on the effects of transient strain accumulation and relaxation resulting from applied mechanical forces during grinding, as well as controllable chemical delamination through capping ligands and oxygen amount during aging. This mechanism features strain-to-defect transformations at the molecular-to-crystal level, which could potentially extend to other 2D metal oxide nanostructure designs.

Mechanochemical-Aging Synthesis of Bismuth Oxide Nanosheets for Photocatalysis

Fe3O4/ZIF-8-90 Nanocomposite as a Strategy for Oncological Treatment

Multidrug-resistant Acinetobacter baumannii (MDR AB) poses a critical global health challenge due to limited treatment options and robust resistance mechanisms. Targeting bacterial bioenergetics, particularly ATP synthase, represents a promising alternative approach inspired by the success of bedaquiline against Mycobacterium tuberculosis.In this study, a library of trisubstituted pyridines was synthesized and evaluated for ATP synthase inhibition in A. baumannii. Four lead compounds demonstrated potent and selective inhibition, exhibiting strong antibacterial activity against both susceptible and MDR clinical isolates. Furthermore, these compounds acted synergistically with colistin, underscoring their potential as novel antibiotics to combat resistant A. baumannii infections.<blockquote><h4>The original article</h4><h5><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380">Synthesis and Evaluation of Pyridine-Based Antibacterial Agents that Inhibit ATP Synthase in Acinetobacter baumannii</a></h5>Angelina L. Dennison, Armaan Singh, Toni A. Marchlewski, Sierra N. Ghee, Kevin C. Gence, lAva G. Hammock, Sabrina Liu, Shaylla Wilson, Alexander P. L. Williams, Katie T. Ward, Thomas E. Meigs, P. Ryan Steed*, and Amanda L. Wolfe*ACS Omega 2025, 10, 39, 45823–45839<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.5c06380">https://doi.org/10.1021/acsomega.5c06380</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Antimicrobial resistance─a leading cause of death globally─was responsible for &gt;4 million premature deaths in 2019, with projections suggesting this number could rise to over 10 million by 2050. <a href="javascript:void(0);">(1,2)</a> The nosocomial ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) contribute significantly to these deaths. Multidrug resistant A. baumannii (MDR AB), an opportunistic Gram-negative pathogen, poses a serious challenge due to a multitude of resistance pathways, including a robust outer membrane (OM), active efflux pumps, and exiguous aperture porins. Moreover, AB is considered one of the most difficult-to-treat ESKAPE pathogens due to its resistance to safety net antibiotics (including colistin, tigecycline, and carbapenems), which demonstrates the dire need for development of novel antimicrobials. <a href="javascript:void(0);">(3)</a>Recently, efforts in antibiotic discovery and development have shifted toward identifying new bacterial targets to overcome current resistance mechanisms in pathogens. One emerging target is ATP synthase, an essential enzyme in bacterial bioenergetics and all life (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>A). <a href="javascript:void(0);">(4−6)</a> F1Fo ATP synthase is a protein complex comprised of two rotary motors that catalyzes the final step in oxidative phosphorylation. <a href="javascript:void(0);">(7)</a> In bacteria, the membrane-embedded Fo motor is composed of stator ab2 subunits adjacent to a homooligomeric ring of c subunits (c10 in AB). <a href="javascript:void(0);">(8)</a> Rotation of the c-ring, driven by an electrochemical gradient of H+, is coupled to rotation of a central stalk within the cytoplasmic F1 motor (composed of α3β3γδε), which catalyzes phosphorylation of ADP to ATP. ATP synthase has proven to be a druggable antibiotic target through the success of the FDA-approved antitubercular drug bedaquiline (BDQ). <a href="javascript:void(0);">(9)</a> BDQ inhibits ATP production in Mycobacterium tuberculosis (MT) by binding to the H+ binding site at the subunit ac interface in Fo, halting ATP synthesis in the F1 complex and causing cell death. <a href="javascript:void(0);">(10−12)</a> BDQ was the first antibiotic to target bacterial energy metabolism and the first drug approved for MDR MT in over four decades. <a href="javascript:void(0);">(13)</a>Due to the success of BDQ against MT, our group <a href="javascript:void(0);">(14−17)</a> and others <a href="javascript:void(0);">(18−21)</a> have begun to explore whether functionalized quinolines can inhibit ATP synthase in other bacterial pathogens via a similar binding interaction with the Fo c subunit and act as antibiotics against resistant strains. One challenge of targeting ATP synthase in Gram-negative pathogens, like AB specifically, is that the Fo complex of ATP synthase is embedded in the inner membrane. Therefore, the drugs must first penetrate the negatively charged OM and then embed in the hydrophobic inner membrane to inhibit the enzyme. Recently, we have developed and interrogated a series of functionalized quinolines that are able to inhibit ATP synthase in AB and penetrate the OM to act as antibiotics against MDR AB strains. <a href="javascript:void(0);">(17)</a> Specifically, the quinoline core was substituted with a benzyl sulfide at the C1 position and a flexible and basic nitrogen-containing side chain at C2. Functionalization on the western portion of the quinoline increased enzyme inhibition and overall antibacterial activity against both susceptible and MDR AB clinical isolates. However, increasing steric bulk beyond a certain threshold decreased both enzymatic and bacterial activity significantly, which was attributed to overall larger steric bulk limiting the interactions with the c subunit and reducing membrane permeability. Quinoline WSA 261 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>B) demonstrated the most potent antibacterial activity against both susceptible and MDR AB of the series. <a href="javascript:void(0);">(17)</a> To explore the necessity of the quinoline core and to interrogate the limitations on molecular flexibility and size on AB ATP synthase inhibition more broadly, we have synthesized a library of 34 novel trisubstituted pyridine analogs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>C) and evaluated this library in enzymatic, antibacterial, and cytotoxicity assays to assess the viability of this series as potential antibacterial agents for treating AB infections.<h4>Methods</h4>Reagents and solvents were purchased reagent-grade and used without further purification. All reactions were performed in flame-dried glassware under an Ar or N2 atmosphere. Evaporation and concentration in vacuo was performed at 40–45 °C. TLC was conducted using precoated SiO2 60 F254 glass plates from EMD with visualization by UV light (254 or 366 nm). NMR (1H or 13C) were recorded on an Varian INOVA-400 MHz spectrometer or a Bruker AVANCE-400 MHz spectrometer at 298 K. Residual solvent peaks were used as an internal reference (CDCl3 with 0.1% TMS). Coupling constants (J) (H,H) are given in Hz. Coupling patterns are designated as singlet (s), doublet (d), triplet (t), multiplet (m) or quintet (qu). IR spectra were recorded on a <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=1&amp;search=IRSpirit">Shimadzu IRSpirit FT-IR spectrophotometer</a> and measured neat. Low-resolution mass spectral data were acquired on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2020">Shimadzu single quadrupole LCMS-2020.</a> High-resolution mass spectral Samples were analyzed with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=3&amp;search=Q+Exactive+HF-X">Q Exactive HF-X</a> (ThermoFisher, Bremen, Germany) mass spectrometer. Samples were introduced via a heated electrospray source (HESI) at a flow rate of 10 μL/min. HESI source conditions were set as: nebulizer temperature 400 °C, sheath gas (nitrogen) 20 arb, auxiliary gas (nitrogen) 0 arb, sweep gas (nitrogen) 0 arb, capillary temperature 320 °C, RF voltage 45 V. The mass range was set to 100–1000 m/z. All measurements were recorded at a resolution setting of 120,000. Solutions were analyzed at 0.1 mg/mL or less based on responsiveness to the ESI mechanism.<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1758"> Xcalibur</a> (ThermoFisher, Breman, Germany) was used to analyze the data. Molecular formula assignments were determined with Molecular Formula Calculator (v 1.3.0). All observed species were singly charged, as verified by unit m/z separation between mass spectral peaks corresponding to the 12C and 13C12Cc-1 isotope for each elemental composition.<h4>Results and Discussion</h4><h5>ATP Synthase Inhibition</h5>Once synthesized, the pyridine compounds were tested for their ability to inhibit ATP synthase activity using our previously reported luciferin/luciferase assay. <a href="javascript:void(0);">(17)</a> Briefly, endogenous electron transport chains of inverted inner membrane vesicles were energized with NADH to generate a proton gradient and to drive ATP synthesis. ATP was measured via luminescence from the luciferase-catalyzed oxidation of d-luciferin. Luminescence activities across increasing inhibitor concentrations were corrected for gradient independent background sources of ATP by comparison with a control containing the protonophore CCCP (carbonyl cyanide 3-chlorophenylhydrazone). The data were then fit to a four-parameter logistic dose–response curve, from which IC50 values and Hill coefficients were determined using a nonlinear least-squares regression.Assessing the SAR of ATP synthase inhibition, pyridines with H or Br at C4 exhibited the lowest inhibitory activity across all amine series (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c06380/suppl_file/ao5c06380_si_001.pdf">Table S1</a>, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig2">Figure 2</a> and <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c06380/suppl_file/ao5c06380_si_001.pdf">S1</a>). Among compounds having C4 phenyl groups, the 4-methoxyphenyl, 3-fluoro-4-methoxyphenyl, and 4-methylphenyl analogs showed potent IC50 values across all amine types, while the 2,4-difluorophenyl, phenyl, and 4-fluorophenyl substituents generally demonstrated weaker inhibitory activity. Compounds WSA 290, 289, 275, 288, and 305 showed the strongest inhibitory activity among the pyridines, with IC50 values between 190 and 270 ng/mL. These in vitro IC50 values are significantly lower than that of WSA 261 (IC50 = 770 ng/mL), suggesting that the pyridine scaffold improves AB ATP synthase inhibitory activity. WSA 290, 289, and 288 all belonged to the C2 cyclopentyl piperidine substituted series, while WSA 275 belonged to the methyl piperazine series and WSA 305 belonged to the ethyl piperidine series. WSA 290 and WSA 275, the strongest performing compounds in their amine series, contained 3-fluoro-4-methoxyphenyl at C4; similarly, WSA 297 (IC50 = 270 ng/mL) with the same 3-fluoro-methoxyphenyl C4 substituent was the strongest performing compound within the benzyl pyrrolidine series. WSA 288 and WSA 305 had methoxyphenyl C4 substituents, and WSA 289 contained a methylphenyl group off C4. The presence of a methoxy group, in both ortho disubstituted and monosubstituted forms, on the C4 phenyl seems to confer stronger inhibition.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_45823_45839_Figure_2_Potent_inhibition_of_ATP_synthesis_activity_86ae703368.jpg" alt="ACS Omega 2025, 10, 39, 45823–45839: Figure 2. Potent inhibition of ATP synthesis activity. ATP synthesis activities of inverted membrane vesicles in the presence of 0–16 μg/mL inhibitor (log2 scale) are plotted relative to the DMSO control containing no inhibitor. Replicate measurements are shown as dots. The fitted dose response curves are shown as solid lines, and the dashed lines indicate the 95% confidence bounds of the fit." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_45823_45839_Figure_2_Potent_inhibition_of_ATP_synthesis_activity_86ae703368.jpg 245w," sizes="100vw" width="4151" height="2101"><h5>Computational Docking</h5>Computational docking was performed using GNINA and PLIP (see <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#sec4">Methods</a>). The protein (PDB ID: <a target="_blank" rel="noopener noreferrer" href="http://doi.org/10.2210/pdb7P2Y/pdb">7P2Y</a>) <a href="javascript:void(0);">(22)</a> was assigned protonation states using the H++ server, <a href="javascript:void(0);">(23)</a> compounds were protonated at pH 7 using Open Babel, <a href="javascript:void(0);">(24)</a> and both were prepared for docking using Autodock Tools. Docking was performed using GNINA with a fixed grid box centered around cAsp60. Poses from GNINA were then input into PLIP 2.3.0 to extract interaction level data.The strong inhibitors WSA 290, 289, 275, 288, and 305 all formed salt-bridges between their secondary or tertiary amines and cAsp60 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c06380/suppl_file/ao5c06380_si_001.pdf">Table S2</a>). The conformations adopted also allowed for hydrogen bonding with the backbones of cPhe53, cGly57 (WSA 290), cMet64 (WSA 275), and aLeu257 (WSA 305). Additionally, cIle65, cLeu71, aIle242, aLeu245, aIle246, and aLeu257 formed a hydrophobic binding pocket that consistently interacts with these compounds. The enriched binding pocket for high affinity compounds is shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig3">Figure 3</a>A, where residue-ligand contacts were computed using PLIP on the chosen GNINA poses for each compound. This binding pocket overlaps substantially with that predicted for our previous quinoline scaffold (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>A), supporting a conserved binding mode centered around the cAsp60-mediated salt-bridge and a hydrophobic cavity across both chemotypes.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_45823_45839_Figure_3_A_The_putative_binding_site_in_the_ac_interface_of_A_baumannii_ATP_synthase_25731daecd.jpg" alt="ACS Omega 2025, 10, 39, 45823–45839: Figure 3. (A) The putative binding site in the ac interface of A. baumannii ATP synthase (PDB ID: 7P2Y) is shown at the interface of subunit a (left, pale cyan) and the c-ring (right, light blue). Residues in the binding pocket are colored by enrichment score, defined here as frequency of binding to strong inhibitors divided by frequency of binding to weak inhibitors, where strong inhibitors are those with IC50 &lt; 500 ng/mL and weak inhibitors are those with IC50 &gt; 5000 ng/mL. The residues shown as sticks are those with enrichment &gt;1 (i.e., frequent interaction with strong inhibitors) and cAsp60 (a key salt-bridge forming residue). (B) Computed binding poses of compounds WSA 276 (cyan), WSA 278 (yellow), and WSA 280 (magenta) in the methyl piperazine series with 4-dimethylamino, 4-methoxyphenyl, and 4-methylphenyl western fronts. (C) Computed binding poses of compounds WSA 288 (cyan), WSA 289 (magenta), and WSA 302 (yellow) in the cyclopentyl piperidine series with 4-methoxyphenyl, 4-methylphenyl, and phenyl western fronts, respectively. (D) Computed binding poses of compounds WSA 297 (yellow), WSA 298 (cyan), WSA294 (orange), and WSA296 (purple) of the benzyl pyrrolidine series, with 2,4-fluoromethoxyphenyl, 4-fluorphenyl, 4-phenyl, and 4-methoxyphenyl C4 substituents respectively." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_45823_45839_Figure_3_A_The_putative_binding_site_in_the_ac_interface_of_A_baumannii_ATP_synthase_25731daecd.jpg 159w," sizes="100vw" width="1868" height="1838"><h4>Conclusions</h4>As discussed, development of new antibiotics that overcome bacterial resistance in MDR AB is a critical need globally due to the rising rate of bacterial infections caused by this pathogen. Bacterial bioenergetics are underexplored drug targets that if properly and selectively inhibited could provide a robust area for antibiotic development, helping to slow the rate of resistance emergence. While the quinoline scaffolds derived from the antitubercular ATP synthase inhibitor BDQ have been the standard for inhibitor development of this type to date, limitations in molecular design and synthesis require new scaffolds to be explored.Through biochemical and antibacterial evaluation of our trisubstituted pyridine library we have demonstrated that the traditional quinoline core is not required for ATP synthase inhibition in AB, opening a new area of drug development from a more readily available starting material. Specifically, we explored the SAR of AB ATP synthase inhibition and found that enzyme inhibition is driven by both molecular size and interaction with key residues in the proposed binding site of the ac interface of Fo. However, despite the increase in enzyme inhibition compared to the traditional quinoline series, antibacterial activity was limited, and the most potent compounds were cytotoxic against mammalian cells indicating that there is room for improvement using strategic molecular design. Four pyridines, WSA 276, WSA 288, WSA 289, WSA 298, with variations at C2 and C4 but similar overall molecular size and flexibility demonstrated potent antibacterial activity against both susceptible and MDR AB clinical isolates due to selective inhibition of AB ATP synthase in ng/mL concentrations. Additionally, this set had the most favorable preliminary toxicity ratio when dosed in combination with colistin, making them the basis for future studies to improve activity and selectivity of the class.

Synthesis and Evaluation of Pyridine-Based Antibacterial Agents that Inhibit ATP Synthase in Acinetobacter baumannii

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