Development of Cyclometalated Iridium(III) Complexes of 2-Phenylbenzimidazole and Bipyridine Ligands for Selective Elimination of Gram-Positive Bacteria.

{"full_text": " doi.org/10.1002/asia.202401060 Research Article\n www.chemasianj.org\n\n\nDevelopment of Cyclometalated Iridium(III) Complexes of\n2-Phenylbenzimidazole and Bipyridine Ligands for\nSelective Elimination of Gram-Positive Bacteria\nAryan Gautam,[a] Ajay Gupta,[a] Puja Prasad,[b] and Pijus K. Sasmal*[a]\n\nHerein, we have reported a series of cationic aggregation- of clinical interest. The complexes Ir2-Ir4 exerted potent\ninduced emission (AIE) active iridium(III) complexes (Ir1-Ir5) of antibacterial activity towards Gram-positive strains with low\nthe type [Ir(C N)2(N N)]Cl, wherein C N is a cyclometalating 2- minimum inhibitory concentrations (MICs) values in the range\nphenylbenzimidazole ligand with varying alkyl chain lengths of 1\u20139 \u03bcM, which is comparable to clinically approved antibiotic\nand N N is a 2,2\u2019-bipyridine ligand attached to bis-polyethylene vancomycin. In contrast, these complexes were found to be\nglycol chains, for the treatment of bacterial infections. The AIE inactive towards Gram-negative bacterial strains (MICs\nphenomenon of the complexes leveraged for detecting bacteria > 100 \u03bcM). The mechanism of antibacterial activity of the\nby fluorescence microscopy imaging that displayed a strong complexes implies that ROS generation, membrane depolariza-\nred emission in Gram-positive bacteria. The antibacterial activity tion and rupture are responsible for bacterial cell death. Further,\nof the complexes assessed against Gram-positive methicillin- the complexes Ir1-Ir3 were found to be low-toxic against\nsensitive S. aureus, methicillin-resistant S. aureus, E. faecium and human red blood cells and human embryonic kidney (HEK293)\nE. faecalis and Gram-negative E. coli and P. aeruginosa bacteria cells, indicating their potential for use as antibacterial agents.\n\n\n\nIntroduction Gram-positive human pathogens that induce a variety of\n infectious diseases. Moreover, the emergence and increase in\nBacterial infections have emerged as a significant global resistance of Gram-positive methicillin-resistant S. aureus\nconcern, impacting millions of people worldwide.[1\u20133] The (MRSA) against many antibiotics leads to a high mortality\ndevelopment of antibiotics have revolutionized medicine, with rate.[8,9] Additionally, MRSA has become a major nosocomial\nthe discovery of penicillin marking the beginning of a new era pathogen in many hospitals worldwide which has become a\nin medicine. However, the rapid development of bacterial growing public health concern.[9,10] Therefore, there is a\nresistance to most of the existing antibiotics poses a serious persuasive demand for the development of novel and potent\npublic health threat. In 2019, over 1.3 million deaths are antibacterial agents for treating S. aureus infections. Over the\nestimated from antimicrobial resistance (AMR), which is bound past few years, significant effort has been made in designing\nto increase if no new antibiotics are discovered.[1\u20135] Medicinal metal complexes for eradicating bacterial infections and\nchemists mainly focus on the modification of existing anti- AMR.[11\u201320] The metal complexes that possess antibacterial\nbiotics, which may only have a limited impact in the short term capabilities have been termed \u201cmetalloantibiotics\u201d. Metalloanti-\nsince pathogens adapt and become resistant to these modified biotics are underexplored compounds that have gained consid-\nantibiotics. Recently, Frei et al. highlighted that approx. 75 % of erable traction as potential antimicrobial agents in recent\nthe antimicrobial agents currently in clinical development are years.[21] Metal complexes with their three-dimensional shapes\nsimply derivatives of already approved antibiotics.[6] This under- provide access to interact with different target sites of bacteria\nscores the importance to design entirely novel classes of and offer a variety of modes of actions such as redox activation,\ncompounds with unique modes of action to combat AMR. ligand exchange or release, reactive oxygen species (ROS)\n The World Health Organization (WHO) has listed ESKAPE generation, catalytic generation of toxic species etc., which\nbacteria as critical and high priority pathogens since they are makes them superiors over organic scaffolds in combating\ninsensitive to common antibacterial treatments.[2,7] Among AMR.[1,6]\nthese, Staphylococcus aureus (S. aureus) is one of the prevalent Recently, metal-based aggregation-induced emission lumi-\n nogens (AIEgens) with metal ions, such as Ir(III),[1,22\u201328] Ru(II),[29]\n[a] A. Gautam, A. Gupta, P. K. Sasmal Pt(II),[30\u201332] Zn(II),[33,34] have shown great promise as antimicrobial\n School of Physical Sciences, Jawaharlal Nehru University, New Delhi 110067, theranostic agents. Among these, cationic cyclometalated Ir(III)\n India AIEgen complexes have earned significant attention due to\n E-mail: pijus@mail.jnu.ac.in\n their various advantages like kinetic inertness, positive charges,\n[b] P. Prasad\n rich photophysical and photochemical properties, and gener-\n Amity Institute of Click Chemistry Research and Studies, Amity University,\n Noida, Uttar Pradesh 201303, India ation of high levels of ROS, which make them promising\n Supporting information for this article is available on the WWW under candidates for detection, imaging and killing of bacterial\n https://doi.org/10.1002/asia.202401060 infections.[1,22\u201326] Recently, our group has reported AIE-active\n\n\nChem Asian J. 2025, 20, e202401060 (1 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\ncationic cyclometalated Ir(III) complexes of 2-phenylquinoline inactive towards Gram-negative E. coli and P. aeruginosa\nand bipyridine ligands that exhibits simultaneous detection and bacteria with > 100 \u03bcM. The mechanistic investigation of\neradication of drug-resistant bacteria.[23] Chen, Wong and co- complexes revealed dual modes of action for antibacterial\nworkers have investigated AIE-active Ir(III) complexes with activity via membrane depolarization and disruption followed\ncyclometalating phenylpyridine and phenylquinoline ligands for by ROS generation.\nfluorescence imaging and photodynamic inactivation of both\nGram-positive and Gram-negative bacteria through ROS\ngeneration.[24] On the other hand, Wang et al. developed AIE- Result and Discussion\nactive cyclometalated Ir(III) photosensitizers for selective detec-\ntion, imaging, and eradication of Gram-positive bacteria.[25] To Design, Synthesis and Characterization of Iridium(III)\ndate, there are reports on Ir(III) complexes with cyclometalating Complexes (Ir1-Ir5)\nphenylquinoline, phenylpyridine, phenylbenzothiazole, and\n(2,4-difluorophenyl)pyridine ligands as bacterial imaging and In this investigation, a series of cationic cyclometalated\ntherapeutic agents.[22\u201326] In contrast, there is only a single report iridium(III) complexes (Ir1-Ir5) were designed and synthesized\nof cyclometalated Ir(III) complexes with biologically important to serve as antibacterial theranostic agents. The iridium(III)\nphenylbenzimidazole ligands as antibacterial agents.[35] For this, centers in the complexes were coordinated with two cyclo-\nRuiz and co-workers have disclosed Ir(III) complexes with metalating biologically important 2-phenylbenzimidazole li-\ncyclometalating 2-phenylbenzimidazole, and N N donor phe- gands and one N N donor 2,2\u2019-bipyridine ligand to achieve an\nnanthroline ligands demonstrating efficient antibacterial activity octahedral geometry (Scheme 1). The 2-phenylbenzimidazole\nselectively against Gram-positive bacteria. The mechanism of ligands in the complexes are attached to alkyl groups of varying\nantibacterial activity of these complexes is mainly due to ROS chains to provide hydrophobic interaction with alkane chains of\ngeneration while without affecting the cell wall and membrane the phospholipid layer and pathogenic components, such as\nor damaging DNA of the bacteria.[35] However, there is no report lipoteichoic acid (LTA) and lipopolysaccharide (LPS), which are\non iridium AIEgens coordinated with 2-phenylbenzimidazole present on the outer cell membranes of Gram-positive and\nligands functionalized with varying lengths of alkyl groups for Gram-negative bacteria, respectively. In contrast, 2,2\u2019-bipyridine\nantibacterial therapy through multiple modes of action. is conjugated with bis-polyethylene glycol chains for better\n In the present study, we have designed and synthesized a aqueous solubility and to minimize the cytotoxicity of these\nseries of cationic AIE-active Ir(III) complexes (Ir1\u2013Ir5) of cyclo- complexes.[23,28] The positive charge on the complexes can\nmetalating 2-phenylbenzimidazole derivatives and N N donor endow electrostatic interaction with negatively charged bacte-\n2,2\u2019-bipyridine ligands as antibacterial theranostic agents rial membranes which would help in detection of bacteria.\n(Scheme 1). A selective detection of Gram-positive bacteria by The benzimidazole ligands (L2\u2013L5) derivatized with different\nthe complexes was observed by fluorescence microscopy alkane chain lengths were synthesized by the reaction of 2-\nimaging, exhibiting strong red emission, corroborating com- phenylbenzimidazole (L1) with bromoalkanes.[36] The iridium-\nplex-mediated bacterial cell aggregation. The complexes Ir2\u2013Ir4 dimers [Ir(Ln)2Cl]2 were synthesized using corresponding benzi-\ndisplayed efficient antibacterial activity against Gram-positive midazole ligands (Ln = L1\u2013L5) with good yields (54\u201378 %). On the\nmethicillin-sensitive S. aureus (MSSA), methicillin-resistant S. other hand, the ancillary BPy-PEG ligand was obtained by the\naureus (MRSA), E. faecium, and E. faecalis with minimum coupling of 2,2\u2019-bipyridine dicarboxylic acid with polyethene\ninhibitory concentrations (MICs) values in the lower micromolar glycol (PEG200).[23,28] The iridium(III) dimers, [Ir(Ln)2Cl]2 were\nrange of 1\u20139 \u03bcM. Whereas, these complexes were completely reacted with BPy-PEG in a mixed solution of DCM-MeOH (2 : 1,\n\n\n\n\nScheme 1. Synthesis of derivatives of 2-phenylbenzimidazole ligands (L1-L5), iridium-dimers ([Ir(Ln)2Cl]2), and cyclometalated iridium(III) complexes (Ir1-Ir5).\nHere, EA = 2-ethoxyethanol; Bpy-PEG = bipyridine polyethylene glycol.\n\n\nChem Asian J. 2025, 20, e202401060 (2 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nv/v) at 50 \u00b0C to afford corresponding iridium(III) complexes (Ir1- coefficients (\u025b) of the complexes are summarized in Table 1. The\nIr5) as illustrated in Scheme 1. The synthesized complexes (Ir1- quantum yields (\u03a6) of the complexes were determined to be in\nIr5) were purified by preparative thin layer chromatography the range of 0.014 to 0.050 (Table 1). Moreover, we assessed the\nand were isolated as chloride salts. The complexes were lipophilicity of our iridium complexes by measuring their\ncharacterized by NMR, ESI-HRMS, and IR. The complexes were octanol (o) /water (w) partition coefficients (Po/w = [C]o/[C]w)\nisolated as a mixture of diastereomers. Detailed synthetic since lipophilicity plays a significant role in the cellular uptake\nprocedures and characterization data for the ligands and of drugs. The determined log Po/w values of the complexes were\ncomplexes are provided in the Experimental Section. The found to be in the range of 1.21 to 1.40 indicating their\ncomplexes showed good solubility in aqueous medium contain- moderately lipophilic nature (Table 1). Notably, the lipophilicity\ning 1 % DMSO. In the solid state, the FT-IR spectra of the of these complexes does not seem to significantly affected by\ncomplexes Ir1-Ir5 showed a strong peak at 1728 cm 1 and a the varying length of the alkane chains on 2-phenylbenzimida-\nstrong but broad peak in the range of 3363\u20133410 cm 1 are zole ligands in the complexes.[37]\nassignable to stretching vibrations of C=O and -OH groups in\nthe Bpy-PEG ligand, respectively. The IR peaks in the range of\n700\u20131585 cm 1 are attributed to the presence of aromatic rings. Determination of AIE Properties of the Iridium(III) Complexes\nThe complexes were structurally characterized by 1H and 13C\nNMR spectra, and the data are presented in the Experimental The AIE properties of the iridium(III) complexes were inves-\nSection and in the Supplementary Information (Figures S1\u2013S15). tigated by measuring emission spectra using fluorescence\nThe mass spectral measurements (ESI-HRMS) exhibited a spectroscopy. Upon excitation at 415 nm, the complexes were\nmolecular ion peak for the ligands (L2\u2013L5) and the complexes weakly emissive in the range of 644\u2013661 nm in aqueous\n(Ir1\u2013Ir5) corresponding to their [M + H] + and [M\u2013Cl ] + species, solution, except for Ir5. The emission intensity of complexes\nrespectively (Figures S16\u2013S24). gradually enhanced with the shift in emission maxima upon the\n addition of THF fraction from 0 to 98 % in the mixed solution\n (Figures 1a and S26a). The concurrent increase in emission\nPhotophysical Properties intensity and shifts in emission maxima of the complexes\n suggest their unique AIE properties.[23,26,28] Next, the aggregation\nThe electronic absorption and emission spectra of the com- properties of the complexes were confirmed by measuring their\nplexes (Ir1\u2013Ir5) were examined in water containing 0.2 % DMSO hydrodynamic size distribution in absence and presence of THF\nat 298 K (Figure S25 and Table 1). The complexes exhibited using dynamic light scattering (DLS) studies (Figures 1 and S26\u2013\nintense absorption bands between 302 and 372 nm, which can S27). For example, the hydrodynamic diameters (d) of Ir2 and\nbe associated to spin-allowed intra-ligand (1IL) \u03c0!\u03c0* transitions. Ir3 in the absence of THF were 59 nm and 79 nm, respectively\nOn the other hand, the broad and weak absorption bands in (Figures 1b and S26b). However, the diameters of Ir2 and Ir3\nthe range of 400 to 526 nm could be attributed to 1MLCT and increased significantly to 459 nm and 396 nm, respectively, in\n3\n MLCT (d\u03c0 (Ir)!\u03c0*) and 1,3LLCT transitions (Figure S25a). These the presence of 25 % THF in the water THF mixture (Figures 1c\ncomplexes displayed a large Stokes\u2019 shifted red emission and S26c). Notably, the hydrodynamic diameter of the com-\nbetween 644 and 661 nm upon excitation at 415 nm (Fig- plexes in water alone is increases with increasing the length of\nure S25b and Table 1). Interestingly, upon increasing the length alkane chains on 2-phenylbenzimidazole ligands, suggesting\nof hydrophobic alkane chains on 2-phenylbenzimidazole ligand that the aggregation capability of the complexes is influenced\nin the iridium complexes, the fluorescence signals significantly by the presence of longer hydrophobic alkane groups (See the\nenhanced. Amongst the complexes studied, complex Ir5 hydrodynamic diameters in the absence and presence of 25 %\nshowed strong fluorescence signals, possibly due to its higher THF in a water-THF mixture in Figure S27).\nextent of self-aggregation capability due to its presence of\nlonger hydrophobic alkane chain and hydrophilic PEG groups.[23]\nThe absorption and emission maxima and molar extinction\n\n\nTable 1. Physicochemical data of the cyclometalated Ir(III) phenylbenzimidazole complexes (Ir1-Ir5).\n Complexes \u03bbabs[a]/nm (\u025b/103 M 1 cm 1) \u03bbem[a]/nm \u03a6[b] log Po/w[c]\n\n Ir1 303 (38.4), 370 (10.1), 400 (6.4), 525 (1.1) 660 0.014 1.34\n Ir2 304 (35.7), 372 (9.6), 402 (6.3), 525 (1.0) 660 0.034 1.32\n Ir3 304 (41.6), 368 (11.2), 400 (7.4), 524 (1.1) 661 0.040 1.31\n Ir4 304 (45.4), 370 (12.4), 402 (7.7), 524 (1.3) 645 0.038 1.21\n Ir5 302 (42.1), 370 (11.4), 402 (6.8), 526 (1.1) 644 0.050 1.40\n\n[a] Absorption (\u03bbabs) and emission (\u03bbem) spectra of the complexes were investigated in water containing 0.2 % DMSO at 298 K. The emission spectra were\nmeasured upon excitation at 415 nm. [b] Quantum yields (\u03a6) were determined in MeCN at 298 K using [Ru(bpy)3](PF6)2 (\u03a6 = 0.0504) as the reference. [c]\nLipophilicity (log Po/w) values determined by measuring the partition coefficient of the complexes in n-octanol/water.\n\n\n\nChem Asian J. 2025, 20, e202401060 (3 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n\n\n\n\nFigure 1. (a) Emission spectra of Ir2 (50 \u03bcM) determined in a water-THF mixture with different THF fractions (ft). Hydrodynamic diameter (d) and particle size\ndistribution of Ir2 (50 \u03bcM) (b) in the absence and (c) in the presence of 25 % THF in a water-THF mixture.\n\n\n\n\nBacteria Imaging Antimicrobial Activity\n\nThe AIE properties of the complexes were utilized for imaging The antimicrobial activities of the iridium(III) complexes (Ir1\u2013Ir5)\nof bacteria. In order to determine the intracellular localization of were evaluated against the ESKAPE pathogens. Among these,\nthe complexes in bacteria, we have used DNA staining Hoechst the Gram-positive bacteria Enterococcus faecium (E. faecium)\ndye. For this, Gram-positive MSSA and Gram-negative E. coli and methicillin-sensitive Staphylococcus aureus (S. aureus)\nbacteria were incubated with 10 \u03bcM of complex Ir2, followed by (MSSA) and the Gram-negative bacteria Pseudomonas aerugino-\nHoechst dye (2 \u03bcM) and then observed under confocal sa (P. aeruginosa) were tested. In addition, Gram-positive\nfluorescence microscope (Figure 2). The imaging results re- methicillin-resistant S. aureus (MRSA) and Enterococcus faecalis\nvealed the selective staining of Gram-positive MSSA bacteria, (E. faecalis) and Gram-negative Escherichia coli (E. coli) were also\nshowing a strong red emission of cells treated with Ir2, examined for clinical interest. Vancomycin (Van) was used as a\nindicating that complex-induced Gram-positive S. aureus bacte- positive control. The activities of the complexes against these\nrial aggregation (Figure 2B). An overlay image of Ir2 and pathogenic bacteria were expressed as minimum inhibitory\nHoechst indicates that DNA is the major target of the complex concentrations (MICs), and compared with clinically approved\n(Figure 2D). However, Gram-negative bacterial staining was also antibiotic vancomycin (Table 2 and Figures S29\u201334). The com-\nobserved only at a higher concentration (200 \u03bcM) of the plexes Ir2\u2013Ir4 exhibited efficient antibacterial activity against\ncomplex (Figure S28). Gram-positive bacteria, with low MIC values 1\u20139 \u03bcM, which is\n comparable to vancomycin and comparable or superior to\n reported metal complexes.[11,12,16\u201320,22\u201325,31\u201335] In contrast, no\n activity of these complexes was determined in Gram-negative\n bacteria (MICs > 100 \u03bcM). This could be attributed to the\n\n\n\n\nFigure 2. Confocal microscopy images of S. aureus and E. coli (108 CFU/ml) bacterial cells treated with 10 \u03bcM of complex Ir2. (A) Bright-field image of bacteria,\n(B) fluorescence image of bacteria treated with Ir2, (C) DNA staining with Hoechst dye, and (D) overlay images of B and C. Scale bar: 5 \u03bcm.\n\n\nChem Asian J. 2025, 20, e202401060 (4 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n\nTable 2. Antibacterial activity against Gram-positive and Gram-negative bacteria and toxicity data in HEK293 cells and human red blood cells of iridium(III)\ncomplexes (Ir1\u2013Ir5).\n Minimum inhibitory concentration (MIC), \u03bcM\n Gram-positive Gram-negative Toxicity (\u03bcM)\n Compounds MSSA MRSA E. faecium E. faecalis E. coli Pa IC50 HC50\n\n Ir1 14.1 13.1 22.2 14.6 > 100 > 100 > 50 > 250\n Ir2 1.0 1.0 1.26 1.5 > 100 > 100 25.2 \ufffd 2.6 > 250\n Ir3 3.0 4.2 4.5 3.5 > 100 > 100 28.0 \ufffd 4.0 > 250\n Ir4 4.7 5.0 8.0 8.8 > 100 > 100 14.6 \ufffd 2.3 160 \ufffd 12\n Ir5 > 25 > 25 > 25 > 25 > 100 > 100 12.9 \ufffd 1.9 172 \ufffd 15\n Van 1.7 1.0 1.0 1.0 75 > 100 - -\n\nMSSA = methicillin-sensitive Staphylococcus aureus; MRSA = methicillin-resistant Staphylococcus aureus; E. faecium = Enterococcus faecium; E. faecalis =\nEnterococcus faecalis; E. coli = Escherichia coli; Pa = Pseudomonas aeruginosa; Van = Vancomycin.\n\n\n\n\ndifference in cell wall structures of Gram-positive and Gram- variety of mechanistic assays were conducted to predict the\nnegative bacteria, since the latter contain an extra protective killing of bacterial cells by these complexes. For mechanistic\nouter membrane, which acts as an effective barrier against studies, we have chosen the complexes Ir1 and Ir2 containing\nantibacterial agents.[18,20,34,38] Nevertheless, dose-dependent cell unsubstituted and alkyl functionalized 2-phenylbenzimidazole\nviability in Gram-positive bacteria was observed that decreases ligands, respectively. The cytoplasmic membrane depolarization\nwith an increase in complex concentrations. Notably, the was monitored using a membrane voltage-sensitive fluorescent\nantibacterial activity of the complexes decreases with an dye DiSC3(5). This cationic dye accumulates in a healthy\nincrease in alkyl chain lengths on cyclometalating 2-phenyl- polarized bacterial cell and leads to its fluorescence quenching.\nbenzimidazole ligands in the complexes. For example, Ir5 with After bacterial membrane disintegration due to stress-induced\nthe longest alkane chain among the series exhibited the lowest depolarization, the dye can diffuse back to the media,\nantibacterial effect (MIC > 25 \u03bcM). This may be due to the recovering its fluorescence at 670 nm.[23,25] The results showed\nhigher extent of self-aggregation of the complexes with the that the cells treated with dye exhibited initial fluorescence\nlonger alkyl chains (Figures 1(b,c), S26(b,c) and S27) that could quenching, however, upon addition of Ir2 showed a sharp\ncause their less interaction with bacterial cells, which can increase in fluorescence intensity compared to control (Fig-\npotentially decrease their antibacterial properties. Therefore, ure 4a). In contrast, the enhancement of fluorescence signals\nthe extent of bacterial aggregation-induced by the complexes was enormously less with the addition of Ir1. These results\nis one of the reasons that could hinder bacterial growth. In suggest that Ir2 can depolarize the bacterial cell membrane\ncontrast, the complex Ir1 (MIC = 13.1\u201322.2 \u03bcM) with unfunction- more efficiently compared to Ir1. This result is consistent with\nalized phenylbenzimidazole ligand displayed lower activity our antibacterial activity data presented in Table 2.\ncompared to Ir2\u2013Ir4. The reasons for low activity of Ir1 have Next, the cytoplasmic membrane permeabilization was\nbeen provided in the following section under mechanistic studied by monitoring the fluorescence intensity of propidium\nstudies. iodide (PI) dye. The PI cannot be internalized in bacterial cells if\n Subsequently, in order to classify the complexes as bacterio- its membrane is not permeabilized/compromised, resulting in\nstatic or bactericidal agents, we investigated the minimum no fluorescent enhancement of this dye being observed.\nbactericidal concentrations (MBCs) of the complexes in Gram- However, this dye can enter into the cytosol of bacteria once\npositive MSSA bacteria (Figure 3). MBC refers to concentrations the cell membrane of bacteria is permeabilized and then\nwhere no bacterial colonies were observed. The antibacterial fluoresces at 617 nm upon binding to DNA.[23,35] Thus, the\nagents can be defined as bactericidal agents if the MBC/MIC change in bacterial cell membrane permeability by our\nratio is \ufffd 4 or bacteriostatic agents if the ratio is > 4. MBC complexes can be measured by determining PI fluorescence\nvalues for Ir1 and Ir2 in MSSA were determined to be 20 \u03bcM signals. It was found that the fluorescence intensity of PI was\nand 2.5 \u03bcM, respectively. Based on the above results, we remarkably enhanced in Ir2 than Ir1, suggesting greater\nconcluded that the complexes Ir1 and Ir2 exhibit bactericidal membrane permeability by the former (Figure 4b). The finding\nactivity against MSSA. Vancomycin (Van) also showed a is also supported by the iridium complexes of unsubstituted 2-\nbactericidal mode of cell death in MSSA. phenylbenzimidazole ligands displayed antibacterial activity\n without affecting the cell walls and membranes of bacteria but\n through ROS generation as reported by Ruiz et al.[35]\nAntibacterial Mechanism In order to evaluate whether ROS is involved in killing\n bacterial cells, a dichlorofluorescein diacetate (DCFH-DA) assay\nThe mechanisms of antibacterial activity of the iridium(III) was performed. DCFH-DA is a cell-permeable nonfluorescent\ncomplexes were investigated in Gram-positive bacteria MSSA. A dye that after oxidation converted to fluorescent dichlorofluor-\n\n\nChem Asian J. 2025, 20, e202401060 (5 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n\n\n\n\nFigure 3. Photographs of colonies of S. aureus on agar plates incubated with different concentrations of complexes (Ir1 and Ir2) and vancomycin (Van).\n\n\n\nescein (DCF), whose fluorescence is measured at 528 nm.[23,39] ruptured.[24] The damage to bacterial cell membrane (shown by\nThe fluorescence intensity of DCF is proportional to the yellow arrows in Figure 5b,d) revealed the antibacterial effect of\nconcentration of ROS. Accordingly, MSSA treated with DCFH- Ir2.\nDA, followed by incubation with Ir1 and Ir2 for different time\npoints. Our results revealed that the fluorescence intensity of\nDCF enhanced over time in comparison to control, confirming Hemolytic Activity and Cytotoxicity\nROS generation (Figure 4c). Notably, the fluorescence intensity\nof Ir2 was greater than Ir1, indicating the former had greater To evaluate the selectivity of the iridium complexes towards\nantibacterial activity than the latter one, which is also good bacterial cells, we have accessed hemolytic activity (HC50) and\nagreement with our antibacterial results (Table 2). Hence, our cytotoxicity (IC50) in human red blood cells (RBC) and human\nantibacterial mechanisms indicate that Ir1 with an unsubsti- embryonic kidney cells (HEK293), respectively (Table 2, Figures 6\ntuted 2-phenylbenzimidazole ligand is inefficient in depolariz- and S35). The complexes Ir4 (HC50, 160 (\ufffd 15) \u03bcM) and Ir5 (HC50,\ning and permeabilizing the cell membrane, and even in 172 (\ufffd 15) \u03bcM) exhibited lower hemolytic activity, while Ir1\u2013Ir3\nproducing high levels of ROS compared to Ir2 functionalized (HC50, > 250 \u03bcM) did not show hemolytic activity (Figure 6).\nwith an alkyl group in the phenylbenzimidazole ligand. We Interestingly, it was observed that the complexes having longer\nhypothesize that these iridium(III) complexes produce ROS alkane chains on 2-phenylbenzimidazole ligands (e. g., Ir4 and\nthrough a Fenton-like reaction.[40\u201342] Furthermore, the events of Ir5) showed slight hemolytic activity. Similarly, the Ir1 was\nbacterial killing mechanisms in these complexes suggest that found to be non-toxic in HEK293 cells (IC50 > 50 \u03bcM), while the\nmembrane depolarization and membrane permeabilization cytotoxicity of Ir2 and Ir3 were 25.2 and 28.0 \u03bcM, respectively,\noccur prior to ROS generation (Figure 4). which is ~25 and 7 times of their MIC values. On the other\n Further, the antibacterial mechanism of the iridium com- hand, Ir4 is relatively toxic towards mammalian cells and its\nplexes was investigated by transmission electron microscopy cytotoxicity (IC50 = 14.6 \u03bcM) is ~2\u20133 times the MICs (4.7\u20138.8 \u03bcM),\n(TEM) analysis to observe the change in bacterial morphology. whereas, Ir5 (MIC > 25 \u03bcM) was found to be toxic towards\nFor this, we recorded the change in morphology of MRSA with mammalian cells (IC50 = 12.9 \u03bcM). Notably, the selectivity of\nand without treatment of complex Ir2. Without treatment of these complexes decreases against bacterial cells upon con-\ncomplex, bacteria showed smooth and intact spherical shape jugation of longer alkane chains on phenylbenzimidazole\n(Figure 5a,c). However, upon treatment with Ir2, the bacteria ligands (Figure S35). Thus, our combined hemolytic and\nlost their original shape and the bacterial membrane\n\n\nChem Asian J. 2025, 20, e202401060 (6 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n\n\n\n\nFigure 4. (a) Estimation of membrane depolarization, (b) membrane permeabilization and (c) ROS production of MSSA treated with the complexes Ir1 (25 \u03bcM)\nand Ir2 (10 \u03bcM) at different time intervals using DiSC3(5), PI and DCFH-DA dyes, respectively. The arrow in graph (a) indicate the addition of complexes at that\npoint. The black, red and blue symbols and bars represent control (no complex), Ir1 and Ir2, respectively.\n\n\n\n\ncytotoxicity results suggest that the complexes Ir1\u2013Ir3 exhibit antibacterial effect of these complexes is comparable to\ngood biocompatibility with mammalian cells. clinically approved antibiotic vancomycin (Van) and better than\n many reported metal complexes. Interestingly, the antibacterial\n activity of these complexes decreases with the increase in the\nConclusions length of the alkyl chains on 2-phenylbenzimidazole ligands in\n the complexes. Among these, Ir2 was found as a best candidate\nIn summary, in this work, we have designed and synthesized a due to its high antibacterial activity (MIC ~1 \u03bcM) and non-toxic\nseries of cationic AIE-active iridium(III) complexes (Ir1\u2013Ir5) towards human red blood cells and healthy human embryonic\nderived from cyclometalating 2-phenylbenzimidazole deriva- kidney cells (HEK293). The mechanistic investigation suggested\ntives and N N donor 2,2\u2019-bipyridine ligands for imaging and dual mode of action of killing bacterial cell via loss of\nkilling of bacteria. The complexes can selectively stain Gram- membrane integrity followed by ROS.\npositive bacteria, showing strong red emission of the Ir(III)\ncomplexes in the bacteria, indicating complex-mediated aggre-\ngation of bacterial cells. The complexes Ir2-Ir4 exhibited potent Experimental Section\nantibacterial activity against Gram-positive methicillin-sensitive\nS. aureus (MSSA), methicillin-resistant S. aureus (MRSA), Enter- Materials\nococcus faecium (E. faecium) and Enterococcus faecalis (E.\n All commercially available solvents were purchased of analytical\nfaecalis), with low MIC values of 1\u20139 \u03bcM. Whereas, the grade and further dried under argon atmosphere before its use. 2-\ncomplexes were completely inactive against Gram-negative Phenylbenzimidazole, 2,2\u2019-bipyridine-4,4\u2019-dicarboxylic acid,\nbacteria Pseudomonas aeruginosa (P. aeruginosa) and Escherichia poly(ethylene glycol) (PEG200), triethylamine, dichlorofluorescein\ncoli (E. coli) upto the tested concentration of 100 \u03bcM. The diacetate (DCFH-DA), 3,3\u2019-dipropylthiadicarbocyanine iodide\n\n\nChem Asian J. 2025, 20, e202401060 (7 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n\n\n\n\nFigure 5. TEM images of MRSA (a and c) without any treatment and (b and d) with treatment of Ir2 (10 \u03bcM) at 30 min. (c) and (d) are enlarged images of the\nred rectangles in (a) and (b), respectively.\n\n\n\n\nFigure 6. (a) Hemolytic activity of complexes (Ir1\u2013Ir5) in human red blood cells (RBCs) at different concentrations. (b) Photographs in the inset and\nquantitative hemolysis of RBCs by Ir2 at different concentrations (25x, 50x, 100x and 250x of MIC). Here, PC = Positive control (Triton X-100) and PBS as the\nnegative control.\n\n\n\n(DiSC3(5)), bovine serum albumin (BSA), propidium iodide (PI) and sourced from Invitrogen. Bacterial strains methicillin-sensitive S.\nTriton X-100 were purchased from Sigma-Aldrich. IrCl3\u00b73H2O, aureus (MTCC 96) and methicillin-resistant S. aureus (MTCC 96) were\npotassium carbonate and bromoalkanes were obtained from Alfa- obtained from Amity University, Noida and E. faecium, E. faecalis, E.\nAesar. 1-Ethyl-3-(3 dimethylaminopropyl)carbodiimide (EDC), 1- coli (MTCC 1302) and P. aeruginosa (ADK-5) were obtained from All\nhydroxybenzotriazole hydrate (HOBt) and glutaraldehyde were India Institute of Medical Sciences (AIIMS), New Delhi. Human\nprocured from Central Drug House (CDH). Thin layer chromatog- embryonic kidney cells (HEK293) were procured from Cell Reposi-\nraphy (TLC) was sourced from Merck, Germany. Nutrient broth and tory, NCCS, Pune. Double distilled water and Milli-Q water (~\nLuria Bertani agar, and vancomycin (Van) hydrochloride obtained 18.8 m\u03a9.cm resistivity) (CDUFBI001, Millipore, USA) were used for\nfrom HiMedia. Trypsin, Dulbecco\u2019s modified Eagle medium (DMEM), the preparation of all the aqueous solutions. The N N donor\nDulbecco\u2019s phosphate-buffered saline (PBS), fetal bovine serum bipyridine polyethylene glycol (Bpy-PEG) ligand was synthesized\n(FBS), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide according to a method reported in the literature.[23,28] The purity of\n(MTT), and penicillin streptomycin solution (pen-strep) were the synthesized iridium(III) complexes (Ir1-Ir5) used for biological\nprocured from Thermo Fischer Scientific. Hoechst 33342 were\n\n\n\nChem Asian J. 2025, 20, e202401060 (8 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nevaluation was found to be \ufffd 95 % by NMR and high resolution MS L3 (Yield: 40.5 mg, 63 %)\nspectra. 1\n H-NMR (500 MHz, CDCl3): \u03b4 (ppm) 7.83 (m, 1H), 7.72 (m, 2H), 7.52\n (d, J = 5.8 Hz, 3H), 7.42 (m, 1H), 7.31 (m, 2H), 4.23 (t, J = 7.6 Hz, 2H),\nMeasurements 1.80 (m, 2H), 1.28 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H). ESI-HRMS (m/z)\n Calcd for C17H18N2 [M + H] + : 251.1543, found: 251.1472.\nTLC was performed on aluminium plates coated with silica gel\nmixed with fluorescent indicator. The purification of synthesized\nligand and complexes were accomplished using 60\u2013120 mesh silica L4 (Yield: 51.4 mg, 72 %)\ngel column chromatography. 1H and 13C NMR spectra were acquired 1\non a Bruker 400 and 500 MHz spectrometers in CDCl3 or DMSO-d6 H-NMR (500 MHz, CDCl3): \u03b4 (ppm) 7.83 (m, 1H), 7.71 (m, 2H), 7.52\nat ambient temperature with tetramethylsilane (TMS) as an internal (m, 3H), 7.42 (m, 1H), 7.31 (m, 2H), 4.22 (t, J = 7.6 Hz, 2H), 1.82 (m,\nstandard. NMR standards used were as follows: (1H-NMR) CDCl3 = 2H), 1.25\u20131.20 (m, 6H), 0.83 (t, J = 6.9 Hz, 3H). 13C-NMR (125 MHz,\n7.260 ppm; DMSO-d6 = 2.50 ppm. (13C-NMR) CDCl3 = 77.00 ppm; CDCl3): \u03b4 (ppm) 153.83, 143.20, 135.68, 130.80, 129.79, 129.45,\nDMSO-d6 = 39.520 ppm. All chemical shifts (\u03b4) are reported in ppm 128.80, 122.75, 122.42, 120.07, 110.22, 44.82, 31.25, 29.82, 26.46,\nrelative to TMS. Spin multiplicities were reported as a singlet (s), 22.56, 14.02. ESI-HRMS (m/z) Calcd for C19H22N2 [M + H] + : 279.1856,\ndoublet (d), triplet (t), quartet (q), doublet of doublets (dd), found: 279.1768.\nmultiplet (m) and broad (br) with coupling constant (J) reported in\nHz. Electrospray ionization high resolution mass spectra (ESI-HRMS)\nwere obtained using a Waters make ESI-MS model synapt G2 high\n L5 (Yield: 53.5 mg, 68 %)\ndefinition mass spectrometry. Fourier transform-Infrared (FT-IR) 1\n H NMR (400 MHz, CDCl3): \u03b4 (ppm) 7.83 (m, 1H), 7.71 (m, 2H), 7.52\nspectra were measured using IR Affinity-1S (Shimadzu, Kyoto, (m, 3H), 7.42 (m, 1H), 7.31 (m, 2H), 4.22 (m, J = 7.7 Hz, 2H), 1.82 (m,\nJapan) FT-IR spectrophotometer equipped with a single reflection 2H), 1.23 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H). ESI-HRMS (m/z) Calcd for\nattenuated total reflectance (ATR) accessory. The IR spectra were C21H26N2 [M + H] + : 307.2169, found: 307.2121.\nrecorded from 4000 to 450 cm 1 using a resolution of 4 cm 1 with\n45 scans. In IR absorption spectra, the shapes and signal intensities\n(height) of peaks (bands) are denoted by the following abbrevia- General Procedure for the Synthesis of Iridium(III) Dimers\ntions: br = broad, vs = very strong, s = strong, m = medium and w = ([Ir(Ln)2Cl]2)\nweak. UV-vis absorption spectra were recorded using a SpectraMax\nM2 plate reader (Molecular Devices) and an Agilent Technologies An oven dried round bottom flask was charged with IrCl3\u00b73H2O\nCarry 100 spectrophotometer, respectively, at 298 K from 800 to (30 mg, 0.100 mmol) and the corresponding cyclometalating C N\n200 nm. Emission spectra and quantum yields were measured on ligand (Ln, 2.2 equivalent). To this, degassed 2-ethoxyethanol/\nEdinburgh Instruments F900 fluorescence spectrophotometer. Dy- deionized water (2 mL; 3 : 1 v/v) was introduced under argon\nnamic light scattering (DLS) was carried out using Zetasizer Nano atmosphere and the resulting mixture was heated to reflux at\nZS90 (Malvern Instrument Ltd., Worcestershire, UK). Transmission 120 \u00b0C for 24 h. Thereafter, the mixture was cooled to room\nelectron microscopy (TEM) images were taken on a JEOL JEM-1400 temperature, and 4 mL water was added to this and further the\nelectron microscope operated at an acceleration voltage of 200 kV. mixture was stirred for 1 h to obtain colored precipitates. The\n precipitate was filtered and washed with deionized water (3x15 mL)\n and ethanol/water (1 : 1), then dried under high vacuum to afford\nGeneral Procedure for the Synthesis of L2-L5[36] corresponding dimers. Due to solubility issues, the dimers [Ir-\n (L1)2Cl]2 (Yield: 44 mg, 71 %) and [Ir(L2)2Cl]2 (Yield: 50 mg, 74 %)\n2-Phenylbenzimidazole (50 mg, 0.257 mmol) and potassium were proceeded to the next step without characterization.\ncarbonate (71.1 mg, 0.514 mmol) were suspended in DMF (1 mL) in\nan oven-dried round bottom-flask under argon atmosphere and\nstirred for 30 min at room temperature. To the above mixture, [Ir(L3)2Cl]2 (Yield: 57 mg, 78 %)\nbromoalkane (1.2 equivalents) was added in a dropwise manner 1\nover a period of 10 min and then the resulting mixture was heated H-NMR (400 MHz, CDCl3): \u03b4 (ppm) 8.39 (d, J = 8.2 Hz, 1H), 7.48 (d,\nat 65 \u00b0C for 18 h. Thereafter, the mixture was cooled at room J = 7.9 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.92 (m, 1H), 6.71 (m, 1H),\ntemperature and followed by diluted with dichloromethane (15 mL) 6.57 (m, 1H), 6.38 (t, J = 7.0 Hz, 1H), 6.01 (d, J = 7.0 Hz, 1H), 4.53 (m,\nthen washed with water (3x15 mL). The combined organic phase 2H), 2.06 (m, 2H), 1.61 (m, 2H), 1.09 (t, J = 7.4 Hz, 3H).\nwas washed with brine and dried over Na2SO4. The organic phase\nwas evaporated on a rotary evaporator to give a pale yellowish oily\ncrude material. The crude material was purified by column [Ir(L4)2Cl]2 (Yield: 42 mg, 54 %)\nchromatography on silica gel using hexane/EtOAc (5 : 1, v/v) as the 1\n H-NMR (500 MHz, CDCl3): \u03b4 (ppm) 8.38 (d, J = 8.2 Hz, 1H), 7.47 (d,\neluent to yield a colorless oily product. J = 7.7 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 6.71 (t,\n J = 7.3 Hz, 1H), 6.57 (t, J = 7.6 Hz, 1H), 6.38 (t, J = 7.5 Hz, 1H), 6.02 (d,\n J = 7.7 Hz, 1H), 4.52 (m, 2H), 2.07 (m, 2H), 1.59 (m, 2H), 1.48\u20131.38 (m,\nL2 (Yield: 33.1 mg, 58 %) 4H), 0.95 (t, J = 7.1 Hz, 3H).\n1\n H-NMR (500 MHz, CDCl3): \u03b4 (ppm) 7.84 (m, 1H), 7.73 (m, 2H), 7.52\n(m, 3H), 7.44 (m, 1H), 7.32 (m, 2H), 4.29 (q, J = 7.3 Hz, 2H), 1.48 (t, J =\n7.3 Hz, 3H). ESI-HRMS (m/z) Calcd for C15H14N2 [M + H] + : 223.1230, [Ir(L5)2Cl]2 (Yield: 50 mg, 59 %)\nfound: 223.1159. 1\n H-NMR (400 MHz, CDCl3): \u03b4 (ppm) 8.39 (d, J = 8.2 Hz, 1H), 7.47 (d,\n J = 7.6 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.92 (t, J = 7.3 Hz, 1H), 6.71 (t,\n J = 7.0 Hz, 1H), 6.57 (t, J = 7.3 Hz, 1H), 6.39 (t, J = 7.1 Hz, 1H), 6.02 (d,\n J = 7.9 Hz, 1H), 4.52 (m, 2H), 2.07 (m, 2H), 1.60 1.33 (m, 10H), 0.91\n (t, J = 6.8 Hz, 3H).\n\n\n\nChem Asian J. 2025, 20, e202401060 (9 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nSynthesis of Ir1 8.3 Hz, 2H), 7.29 (m, 1H), 7.10 (m, 1H), 6.92 (t, J = 7.8 Hz, 2H), 6.85 (t,\n J = 7.1 Hz, 2H), 6.27 (d, J = 7.0 Hz, 2H), 5.62 (d, J = 8.2 Hz, 2H), 4.69\nA mixture of iridium(III) dimer ([Ir(L1)2Cl]2: 24.5 mg, 0.020 mmol) and (m, 4H), 4.07 (m, 8H), 3.75\u20133.60 (m, 21H), 2.01 (m, 4H), 1.32 (m, 12H),\nN N donor ligand (Bpy-PEG: 26.7 mg, 0.044 mmol) was heated in a 0.88 (d, J = 7.1 Hz, 6H). IR (ATR) \u03bd (cm 1): 3392 br (O H), 2923 m\n25 mL oven-dried round bottom-flask sealed with a rubber septum (C H: aliphatic), 1728 vs (C=O), 1629 w, 1583 w, 1502 m, 1404 w,\ncontaining a mixed solution of CH2Cl2/MeOH (2 : 1, v/v, 2.5 mL) at 1259 s, 1068 s, 1031 m, 742 vs, 702 m. ESI-HRMS (m/z) Calcd for\n50 \u00b0C under argon atmosphere for 6 h. Subsequently, the solution C66H82IrN6O12 + [M\u2013Cl ] + : 1343.5614, found: 1343.6488.\nwas cooled to room temperature, and solvent was removed on a\nrotary evaporator. The resulting solid was washed with hexane\n(3x15 mL). The crude material was purified by preparative thin layer Synthesis of Ir5\nchromatography using CH2Cl2/MeOH (20 : 1, v/v). The product was\ndried under vacuum to afford Ir1 (Yield: 22.7 mg, 47 %). This complex was synthesized in an identical manner to that\n1\n described for Ir1, using a mixture of [Ir(L5)2Cl]2 (33.5 mg,\n H-NMR (500 MHz, DMSO-d6): \u03b4 (ppm) 9.24 (m, 2H), 8.36 (m, 1H), 0.020 mmol) and Bpy-PEG (26.7 mg, 0.044 mmol). Yield: 31 mg,\n8.18 (m, 2H), 7.97 (d, J = 7.5 Hz, 2H), 7.79 (m, 1H), 7.61 (m, 3H), 7.41 54 %.\n(d, J = 7.6 Hz, 1H), 7.22 (m, 2H), 7.05 (t, J = 7.4 Hz, 2H), 6.93 (m, 2H),\n 1\n6.85 (t, J = 7.2 Hz, 2H), 6.20 (d, J = 7.3 Hz, 1H), 5.71 (m, 1H), 4.49 (m, H-NMR (500 MHz, CDCl3): \u03b4 (ppm) 9.16 (m, 1H), 8.91 (m, 1H), 8.31\n4H), 3.81 (m, 4H), 3.72\u20133.37 (m, 24H). 13C-NMR (125 MHz, CDCl3): \u03b4 (m, 2H), 8.09 (m, 2H), 7.77 (t, J = 8.1 Hz, 2H), 7.46 (t, J = 8.2 Hz, 2H),\n(ppm) 163.96, 163.25, 156.88, 152.25, 149.51, 139.53, 139.39, 139.19, 7.29 (t, J = 6.7 Hz, 2H), 7.09 (m, 2H), 6.92 (m, 2H), 6.84 (t, J = 7.3 Hz,\n133.98, 133.27, 132.44, 130.60, 128.01, 124.51, 123.91, 123.83, 2H), 6.27 (d, J = 7.3 Hz, 2H), 5.64 (m, 2H), 4.74\u20134.55 (m, 8H), 4.01 (m,\n123.38, 122.49, 122.38, 113.17, 72.38, 72.33, 72.31, 72.29, 69.77, 4H), 3.73\u20133.51 (m, 25H), 2.01 (m, 4H), 1.50\u20131.27 (m, 20H), 0.86 (t, J =\n68.05, 65.54, 60.21. IR (ATR) \u03bd (cm 1): 3394 br (O H), 2922 m (C H: 7.0 Hz, 6H). 13C-NMR (125 MHz, CDCl3): \u03b4 (ppm) 162.33, 157.55,\naliphatic), 1728 vs (C=O), 1593 m, 1537 m, 1467 m, 1454 m, 1406 w, 157.31, 152.45, 138.84, 135.26, 133.63, 133.57, 131.01, 125.22,\n1257 s, 1232 s, 1101 s, 1022 s, 819 w, 738 vs, 702 w. ESI-HRMS (m/z) 125.14, 124.84, 124.76, 124.06, 123.31, 113.68, 113.55, 111.25,\nCalcd for C54H58IrN6O12 + [M\u2013Cl ] + : 1175.3736, found: 1175.3588. 111.16, 73.09, 72.68, 70.66, 70.58, 70.52, 70.09, 61.86, 61.68, 54.01,\n 45.32, 31.82, 29.82, 29.72, 29.31, 29.24, 26.92, 22.70, 14.20. IR (ATR) \u03bd\n (cm 1): 3410 br (O H), 2922 m (C H: aliphatic) 1728 vs (C=O), 1583\nSynthesis of Ir2 w, 1556 w, 1406 m, 1321 m, 1257 s, 1234 m, 1120 s, 1029 m, 925 w,\n 723 s, 702 w. ESI-HRMS (m/z) Calcd for C70H90IrN6O12 + [M\u2013Cl ] + :\nThis complex was synthesized in an identical manner to that 1399.6240, found: 1399.5513.\ndescribed for Ir1, using a mixture of [Ir(L2)2Cl]2 (26.8 mg,\n0.020 mmol) and Bpy-PEG (26.7 mg, 0.044 mmol). Yield: 24.8 mg, Notably, among the complexes Ir1-Ir5, we have provided 13C NMR\n49 %. data for Ir1 and Ir5. The reason for this is that the iridium center in\n1\n Ir1 and Ir5 is coordinated with unsubstituted 2-phenylbenzimida-\n H-NMR (500 MHz, CDCl3): \u03b4 (ppm) 9.12 (s, 2H), 8.34 (d, J = 5.6 Hz, zole and C8H17 alkyl functionalized 2-phenylbenzimidazole ligands,\n2H), 8.07 (dd, J = 5.6, 1.5 Hz, 2H), 7.82 (d, J = 7.9 Hz, 2H), 7.53 (d, J = respectively. In Ir2-Ir5, 2-phenylbenzimidazole ligands are attached\n8.3 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.11 (m, 2H), 6.92 (t, J = 7.8 Hz, to varying lengths of alkyl groups. In 13C NMR spectra, these alkyl\n2H), 6.86 (m, 2H), 6.28 (d, J = 7.6 Hz, 2H), 5.63 (d, J = 8.3 Hz, 2H), 4.81 carbons overlap and appear almost with identical chemical shift\n(m, 4H), 4.06 (m, 8H), 3.74\u20133.59 (m, 23H), 1.65 (t, J = 7.3 Hz, 6H). IR values. Hence, these alkyl carbons in Ir2-Ir5 are not clearly\n(ATR) \u03bd (cm 1): 3363 br (O H), 2920 m (C H: aliphatic), 1728 vs distinguished from 13C NMR.\n(C=O), 1583 w, 1506 m, 1479 w, 1433 s, 1261 s, 1236 s, 1195 m,\n1105 s, 742 vs, 702 m. ESI-HRMS (m/z) Calcd for C58H66IrN6O12 + [M\u2013\nCl ] + : 1231.4362, found: 1231.5392. UV-Visible Spectra\n The absorption spectra of the iridium(III) complexes Ir1\u2013Ir5 were\nSynthesis of Ir3 determined in water containing 0.2 % DMSO at room temperature.\n All UV-vis spectroscopic measurements were performed in a quartz\nThis complex was synthesized in an identical manner to that cuvette with an optical path length of 10 mm. The wavelength was\ndescribed for Ir1, using a mixture of [Ir(L3)2Cl]2 (29 mg, 0.020 mmol) reported in nanometers (nm).\nand Bpy-PEG (26.7 mg, 0.044 mmol). Yield: 25.9 mg, 49 %.\n1\n H-NMR (400 MHz, CDCl3): \u03b4 (ppm) 9.24 (m, 1H), 8.89 (m, 1H), 8.31\n(m, 2H), 8.03 (m, 2H), 7.78 (d, J = 7.6 Hz, 2H), 7.48 (m, 2H), 7.30 (m, Emission Spectra and Determination of Quantum Yields\n2H), 7.09 (m, 2H), 6.94 (m, 2H), 6.84 (t, J = 7.4 Hz, 2H), 6.27 (m, 2H), Emission spectra of iridium(III) complexes were measured in water\n5.64 (m, 2H), 4.75\u20134.50 (m, 8H), 3.99 (m, 4H), 3.75\u20133.47 (m, 24H), 2.0 containing 0.2 % DMSO at room temperature using a quartz cuvette\n(m, 4H), 1.50 (m, 4H), 1.00 (t, J = 7.3 Hz, 6H). IR (ATR) \u03bd (cm 1): 3405 with an optical path length of 10 mm. The excitation wavelength of\nbr (O H), 2927 m (C H: aliphatic), 1728 vs (C=O), 1583 w, 1556 w, all the complexes was 415 nm. Quantum yields (\u03a6) of all the\n1406 m, 1361 m, 1255 s, 1234 m, 928 m, 759 m, 721 s. ESI-HRMS (m/ complexes were studied in MeCN at room temperature using\nz) Calcd for C62H74IrN6O12 + [M\u2013Cl ] + : 1287.4988, found: 1287.2275. [Ru(bpy)3](PF6)2 (\u03a6 = 0.0504) as the reference.\n\n\nSynthesis of Ir4 Aggregation-Induced Emission Studies[23,26,28]\nThis complex was synthesized in an identical manner to that The iridium complexes (50 \u03bcM) were prepared in water containing\ndescribed for Ir1, using a mixture of [Ir(L4)2Cl]2 (31.3 mg, 1 % DMSO in final volume of 2 ml. The complexes were titrated\n0.020 mmol) and Bpy-PEG (26.7 mg, 0.044 mmol). Yield: 28 mg, with the different amounts of THF keeping the total volume (water\n51 %. plus THF) constant (2 ml) and measured their phosphorescence\n1\n H-NMR (500 MHz, CDCl3): \u03b4 (ppm) 9.20 (s, 2H), 8.32 (d, J = 5.6 Hz, emission spectra. The hydrodynamic diameters (d) of the complexes\n2H), 8.06 (dd, J = 5.6, 1.5 Hz, 2H), 7.78 (d, J = 7.8 Hz, 2H), 7.48 (d, J =\n\n\nChem Asian J. 2025, 20, e202401060 (10 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nin water and its aggregated particles in water-THF mixtures were TEM Imaging of Bacteria[16,28]\ndetermined by DLS.\n A 108 CFU/ml bacterial suspension of MRSA was incubated with\n complex Ir2 (10 \u03bcM) for 30 min at 37 \u00b0C. Subsequently, the bacterial\nDetermination of Lipophilicity suspensions were centrifuged at 4000 rpm for 5 min and washed\n with PBS (pH 7.4) three times. The bacteria were then fixed with\nThe lipophilicity of the iridium(III) complexes were determined by 2.5 % glutaraldehyde in PBS at 4 \u00b0C for 4 h. Further, the bacterial\nmeasuring their partition coefficients (log Po/w) between n-octanol suspensions were centrifuged at 2000 rpm for 5 min and washed\n(o) and water (w) using the classical flask-shaking method.[43,44] with PBS two times, and then centrifuged. Thereafter, the bacterial\nBriefly, 0.5 mg of complexes was taken in 3 ml of n-octanol and pellet was resuspended in 40 % ethanol and dehydrated with 70, 80\nwater mixture (1 : 1, v/v) and mixed vigorously for 24 h. Then the and 90 % ethanol for 30 min each at 4 \u00b0C. 10 \u03bcl of aliquots from\nmixture was kept at room temperature in a stationary state for an sample were deposited on carbon coated copper grids (CF 200 CU,\nadditional 24 h to reach saturation. After that the n-octanol and Electron Microscopy Sciences, USA) and allowed for slow evapo-\nwater phases were separated. The concentration of complexes was ration at room temperature overnight. The dried sample was\nthen measured by UV-vis spectroscopy in both the n-octanol (Co) imaged using a JEOL JEM-1400 electron microscope.\nand water (Cw) phases to determine the log Po/w = log [C]o/[C]w,\nwhere [C]o and [C]w are the concentrations of the complex in the n-\noctanol and water phases, respectively. Mechanistic Studies\n\n ROS Quantification Assay\nBacteria Imaging\n The production of ROS by the complexes was determined using\nMSSA and E. coli bacteria were grown in their mid-log phase and\n dichlorofluorescein diacetate (DCFH-DA) dye by fluorescence spec-\n108 CFU/ml of bacteria were harvested, washed and resuspended in\n troscopy. For this, 108 CFU/ml of MSSA were grown in mid-log\nPBS. Then, 10 \u03bcM of complex Ir2 was incubated with 1 ml of\n phase, then harvested, washed and resuspended in fresh broth. The\nbacterial suspension for 30 min at 37 \u00b0C, and the unbounded Ir2\n stock solution of the complexes was prepared in broth media. The\nwas removed by centrifugation at 3000 rpm for 3 min and the\n bacterial suspension (108 CFU/ml) was treated with the complexes\nbacterial pellet was resuspend in 500 \u03bcl PBS. Subsequently, the\n Ir1 (25 \u03bcM) and Ir2 (10 \u03bcM) in 96 well plates. To this, 5 \u03bcl of DCFH-\nbacteria were stained with nucleic acid dye Hoechst (2 \u03bcM dissolved\n DA was added to reach a final concentration of 5 \u03bcM in a total\nin PBS) for 10 min at room temperature. After this the bacteria were\n volume of 200 \u03bcl. Subsequently, the bacteria in 96 well plates were\nwashed three times with PBS and the bacterial cells were further\n incubated without shaking at 37 \u00b0C and the ROS production was\nresuspended in 100 \u03bcl of PBS. Thereafter, 20 \u03bcl aliquot of bacterial\n monitored by determining the fluorescence intensity of dichloro-\nsuspension were dropped onto clean microscopic slides and\n fluorescein (DCF) at 528 nm (\u03bbex = 488 nm) using SpectraMax M2\ncovered with coverslips for immobilization. Then the stained cells\n plate reader. Bacteria treated with dye alone served as a control.\nwere visualized under Nikon Eclipse Ti E microscope and Magnus\nTrinocular MLXi Plus microscope. Hoechst and complex Ir2 were\nboth excited with 405 nm laser. Cytoplasmic Membrane Depolarization Assay\n Cytoplasmic membrane depolarization of bacteria by the com-\nAntibacterial Activity Studies plexes were monitored by a membrane voltage-sensitive fluores-\n cent dye DiSC3(5). Accordingly, 108 CFU/ml of MSSA bacteria was\nThe antibacterial activity (MIC values) of the complexes against\n grown in mid log phase and then harvested and resuspended in\nvarious Gram-positive and Gram-negative bacteria were evaluated\n Nutrient broth supplemented with 0.5 mg/ml of BSA. BSA solution\nby the broth microdilution method according to CLSI guidelines.[45]\n was added to the broth to reduce DiSC3(5) absorption onto the\nThe bacterial strains were cultured in Nutrient broth to achieve\n polystyrene surface. The stock solutions of the complexes were\n108 CFU/ml confluency. To investigate the MIC, the bacterial density\n prepared in broth media. The bacterial solution was diluted to give\nwas further diluted to 105 CFU/ml. Accordingly, the different\n 107 CFU/ml bacteria. Then 190 \u03bcl of bacterial suspension was\nconcentrations of the compounds were prepared in broth. Then the\n treated with 5 \u03bcl of DiSC3(5) dye (1 \u03bcM final concentration) in 96\n100 \u03bcl media solutions with bacteria were dispensed to each well of\n well plate. The fluorescence of the dye was monitored using\n96 plates. Subsequently, 100 \u03bcl complex solutions were added to\n SpectraMax M2 plate reader at 670 nm (\u03bbex = 610 nm). At first, the\neach well of 96 plates to make the final volume of 200 \u03bcl. The\n dye was equilibrated with the bacteria to obtain maximum self-\ncompound concentrations used in Gram-positive and Gram-\n quenching (or a minimum steady fluorescence signal). Thereafter,\nnegative bacteria in the range of 0.1\u201350 \u03bcM and 1\u2013100 \u03bcM,\n 5 \u03bcl of complex was added to each well at a final concentration of\nrespectively. Then the plates were incubated at 37 \u00b0C for 18 h. The\n 25 and 10 \u03bcM for Ir1 and Ir2, respectively, and the fluorescence\nMIC values were determined by measuring the absorbance at\n signal was monitored for 2 h. A control experiment was performed\n600 nm using SpectraMax M2 plate reader (Molecular Devices). The\n with bacteria suspension treated with dye only.\ninhibition of bacterial growth was calculated with reference to\ncontrol having only bacteria without drug treatment. Minimum\nbactericidal concentrations (MBCs) of the complexes were deter- Cytoplasmic Membrane Permeabilization Assay\nmined by taking 5 \u03bcl volumes of different concentrations of sample\nsolution from the 96-wells and spreading them over agar plates. A 108 CFU/ml of MSSA were grown in mid-log phase and then\nNext, the agar plates were incubated at 37 \u00b0C for 18 h without harvested, washed and resuspended in 1 : 1 ratio of 5 mM glucose\nshaking. The concentration where no visible bacterial colonies were and 5 mM HEPES buffer (pH 7.4). The stock solutions of the\nobserved in the agar plate corresponded to the MBC. Triplicate complexes were prepared in broth media. A 10 \u03bcl of complex\nwells were used for each concentration, and the experiments were solution was then added to 165 \u03bcl of resuspended bacteria in black\nconducted three times. nunc 96 well microtitre plates containing 25 \u03bcl of 10 \u03bcM of\n propidium iodide (PI). The final concentration of iridium complex\n was 25 and 10 \u03bcM for Ir1 and Ir2, respectively, in this total volume\n\n\nChem Asian J. 2025, 20, e202401060 (11 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f 1861471x, 2025, 2, Downloaded from https://aces.onlinelibrary.wiley.com/doi/10.1002/asia.202401060 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nof 200 \u03bcl. The fluorescence intensity of the PI dye was recorded for desh and AIIMS, New Delhi are acknowledged for providing\n2 h at 5 min intervals at an excitation and emission wavelengths of bacterial strains.\n535 nm and 617 nm, respectively. A control experiment was\nperformed with bacteria suspension treated with dye only.\n\n Conflict of Interests\nToxicity Studies\n The authors declare no conflict of interest.\nHemolysis Assay\nThe hemolytic activity of the complexes was tested in human red\nblood cells (RBCs) according to literature report.[11,20] Briefly, 1.5 ml Data Availability Statement\nof human blood was collected in K2EDTA tube from healthy donor\nand centrifuged at 2000 rpm for 10 min to separate the RBCs from The data that support the findings of this study are available\nplasma. Then the RBCs pellet was washed three times with 1XPBS from the corresponding author upon reasonable request.\n(pH 7.4) by centrifuging at 3000 rpm for 3 min. The RBCs pellet was\nresuspended in 10 ml PBS and then the cells were treated with\ndifferent concentrations of the complexes and 10 % Triton X-100 Keywords: Aggregation-induced emission \u00b7 Antibiotics \u00b7 Gram-\nused as positive control. The RBCs suspension was incubated at positive bacteria \u00b7 Iridium \u00b7 Phenylbenzimidazole\n37 \u00b0C for 1 h and then the mixture was centrifuged at 2000 rpm for\n5 min. The supernatant was transferred to a 96 well plate (100 \u03bcl) at\nits absorbance was determined at 540 nm. The hemolysis (%) was [1] P. Prasad, A. Gupta, P. K. Sasmal, Chem. Commun. 2021, 57, 174\u2013186.\ncalculated using the following formula: [2] G. Mancuso, A. Midiri, E. Gerace, C. Biondo, Pathogens 2021, 10, 1310.\n [3] C. J. L. Murray, K. S. Ikuta, F. Sharara, L. Swetschinski, G. Robles Aguilar,\n A. Gray, C. Han, C. Bisignano, P. Rao, E. Wool, S. C. Johnson, A. J. Browne,\nhemolysis \u00f0%\u00de \u00bc \u00bd\u00f0A A0 \u00de=\u00f0Atotal A0 \u00de\ufffd \ufffd 100: M. G. Chipeta, F. Fell, S. Hackett, G. Haines-Woodhouse, B. H. Kashef\n Hamadani, E. A. P. Kumaran, B. McManigal, S. Achalapong, R. Agarwal, S.\nWhere, the absorbance of a well-treated with a complex (A) to that Akech, S. Albertson, J. Amuasi, J. Andrews, A. Aravkin, E. Ashley, F.-X.\nwhich was not treated (A0), and the overall response was Babin, F. Bailey, S. Baker, B. Basnyat, A. Bekker, R. Bender, J. A. Berkley,\n A. Bethou, J. Bielicki, S. Boonkasidecha, J. Bukosia, C. Carvalheiro, C.\nnormalized with respect to the Triton X-treated well (Atotal)\n Casta\u00f1eda-Orjuela, V. Chansamouth, S. Chaurasia, S. Chiurchi\u00f9, F.\nassuming 100 % lysis of RBCs. Chowdhury, R. Clotaire Donatien, A. J. Cook, B. Cooper, T. R. Cressey, E.\n Criollo-Mora, M. Cunningham, S. Darboe, N. P. J. Day, M. De Luca, K.\n Dokova, A. Dramowski, S. J. Dunachie, T. Duong Bich, T. Eckmanns, D.\nCytotoxicity Assay Eibach, A. Emami, N. Feasey, N. Fisher-Pearson, K. Forrest, C. Garcia, D.\n Garrett, P. Gastmeier, A. Z. Giref, R. C. Greer, V. Gupta, S. Haller, A.\nThe cytotoxicity (IC50) of the complexes were evaluated in human Haselbeck, S. I. Hay, M. Holm, S. Hopkins, Y. Hsia, K. C. Iregbu, J. Jacobs,\nembryonic kidney cells (HEK293) by MTT assay. The cells were D. Jarovsky, F. Javanmardi, A. W. J. Jenney, M. Khorana, S. Khusuwan, N.\ncultured in DMEM media with 10 % FBS and 1 % pen-strep at 37 \u00b0C Kissoon, E. Kobeissi, T. Kostyanev, F. Krapp, R. Krumkamp, A. Kumar, H.\nin a CO2 incubator. After 90 % confluency, cells were trypsinized, H. Kyu, C. Lim, K. Lim, D. Limmathurotsakul, M. J. Loftus, M. Lunn, J. Ma,\ncentrifuged and redispersed in complete media. Then the cultured A. Manoharan, F. Marks, J. May, M. Mayxay, N. Mturi, T. Munera-Huertas,\n P. Musicha, L. A. Musila, M. M. Mussi-Pinhata, R. N. Naidu, T. Nakamura,\ncells were seeded in 96-well plate as triplicate at a density of 9000\n R. Nanavati, S. Nangia, P. Newton, C. Ngoun, A. Novotney, D.\ncells/well and the plates were kept in a CO2 incubator for 24 h. The Nwakanma, C. W. Obiero, T. J. Ochoa, A. Olivas-Martinez, P. Olliaro, E.\nstock solution of the complexes was prepared in complete media. Ooko, E. Ortiz-Brizuela, P. Ounchanum, G. D. Pak, J. L. Paredes, A. Y.\nThe media from HEK293 cells were removed and added different Peleg, C. Perrone, T. Phe, K. Phommasone, N. Plakkal, A. Ponce-de-Leon,\nconcentration of complexes from 2.5 to 50 \u03bc\u039c in a 200 \u03bcl of total M. Raad, T. Ramdin, S. Rattanavong, A. Riddell, T. Roberts, J. V.\nvolume. The cells were incubated with the complexes for 24 h in a Robotham, A. Roca, V. D. Rosenthal, K. E. Rudd, N. Russell, H. S. Sader, W.\nCO2 incubator. The cells were then washed with PBS followed by Saengchan, J. Schnall, J. A. G. Scott, S. Seekaew, M. Sharland, M.\naddition of 200 \u03bcl of DMEM and 20 \u03bcl of MTT solution (5 mg ml 1 in Shivamallappa, J. Sifuentes-Osornio, A. J. Simpson, N. Steenkeste, A. J.\n Stewardson, T. Stoeva, N. Tasak, A. Thaiprakong, G. Thwaites, C. Tigoi, C.\nPBS) to each well and incubated at 37 \u00b0C for 3 h. Then the medium\n Turner, P. Turner, H. R. van Doorn, S. Velaphi, A. Vongpradith, M.\nwas discarded and replaced with 200 \u03bcl of DMSO in each well to Vongsouvath, H. Vu, T. Walsh, J. L. Walson, S. Waner, T. Wangrangsima-\ndissolve the formazan crystals formed. The cell viability was kul, P. Wannapinij, T. Wozniak, T. E. M. W. Young Sharma, K. C. Yu, P.\ndetermined by measuring the absorbance at 570 nm using a Zheng, B. Sartorius, A. D. Lopez, A. Stergachis, C. Moore, C. Dolecek, M.\nSpectraMax M2 microtitre plate reader. Naghavi, Global burden of bacterial antimicrobial resistance in 2019: a\n systematic analysis. Lancet 2022, 399, 629\u2013655.\n [4] T. Coque, D. Graham, A. Pruden, A. So, E. Topp, Bracing for Superbugs:\n Strengthening environmental action in the One Health response to\n antimicrobial resistance, United Nations Environment Programme,\nAcknowledgements Technical report, 2023.\n [5] J. O\u2019Neill, Antimicrobial resistance: Tackling a crisis for the health and\n wealth of nations, Rev. Antimicrob. Resist. 2014, 20, 1\u201316 .\nP. K. S. acknowledges MHRD-STARS (MoE-STARS/STARS-1/374), [6] A. Frei, J. Zuegg, A. G. Elliott, M. Baker, S. Braese, C. Brown, F. Chen, C. G.\nBRNS (58/14/15/2023-BRNS/12127), and SERB, DST (CRG/2023/ Dowson, G. Dujardin, N. Jung, A. P. King, A. M. Mansour, M. Massi, J.\n005243) for financial support. P. P. acknowledge SERB, DST Moat, H. A. Mohamed, A. K. Renfrew, P. J. Rutledge, P. J. Sadler, M. H.\n Todd, C. E. Willans, J. J. Wilson, M. A. Cooper, M. A. T. Blaskovich, Chem.\n(SRG/2023/001099) for financial support. A. Gautam and A. Sci. 2020, 11, 2627\u20132639.\nGupta gratefully acknowledge the BRNS and MHRD-STARS, [7] E. Tacconelli, E. Carrara, A. Savoldi, S. Harbarth, M. Mendelson, D. L.\nrespectively, for fellowships. We thank AIRF, JNU for the Monnet, C. Pulcini, G. Kahlmeter, J. Kluytmans, Y. Carmeli, M. Ouellette,\n K. Outterson, J. Patel, M. Cavaleri, E. M. Cox, C. R. Houchens, M. L.\ninstrumentation facilities. AICCRS, Amity University, Uttar Pra- Grayson, P. Hansen, N. 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Wang, ACS Materials Accepted manuscript online: November 9, 2024\n Lett. 2023, 5, 162\u2013171. Version of record online: November 23, 2024\n\n\n\n\nChem Asian J. 2025, 20, e202401060 (13 of 13) \u00a9 2024 Wiley-VCH GmbH\n\f", "pages_extracted": 13, "text_length": 92741}