High-throughput triazole-based combinatorial click chemistry for the synthesis and identification of functional metal complexes

Domain: chemistry
Published: 2025-12-23
Editorial outcome: minor_revision

Abstract
Transition metal complexes have found many applications in the modern world ranging from catalysts, luminescent materials to bioactive compounds and more. However, methods for the systematic synthesis and evaluation of large numbers of metal compounds at a time are still limited. Here, a high-throughput combinatorial approach using copper(I)-catalyzed alkyne–azide cycloaddition chemistry is implemented to make 192 bidentate pyridyl-1,2,3-triazole ligands. This ligand library is coordinated to five metal scaffolds under mild conditions to yield 672 metal compounds, allowing for an accelerated exploration of the transition metal complex chemical space. The prepared libraries are showcased for compound discovery in antimicrobial applications through a “Direct-to-Biology” approach, with selected libraries further tested for catalytic and photophysical properties. Six promising metalloantibiotics are identified and re-synthesized, exhibiting activity against Gram-positive bacteria in the nanomolar range with favorable therapeutic windows. Two iridium complexes are selected from a transfer hydrogenation screening assay and re-synthesized, with one showing high catalytic activity. This methodology represents a significant step forward in the field of metal-based combinatorial chemistry and its application towards the systematic coverage of vast chemical spaces in the search for molecules with optimized properties.

Introduction
The Copper(I)-catalyzed Alkyne–Azide cycloaddition (CuAAc Click) reaction to form substituted 1,2,3-triazoles (hereafter referred to as “triazoles”) is one of the cornerstones of modern chemistry1–4. Functional group tolerance, bio-compatible coupling conditions and high yields make it a powerful transformation. Although mostly used as a convenient way of linking molecular fragments together, it is possible for the resulting triazole unit to bind to metal centers as a ligand. This coordination opens up avenues for catalysis and complexes with highly tunable photophysical properties5,6. Incorporation of a pyridine or other lone pair donating group to the triazole compound enables formation of bidentate ligands, which are able to chelate metal centers effectively via 5- and 6-membered metallocycles (Fig. 1)7. The binding modes of these ligands can be varied by switching the substitution site on the triazole ring (through different pyridine/azide structures), giving ligands with substantially different chemistries8. Coordination tends to occur through the more electron rich N3, giving more stable complexes, while coordination through the more electron deficient N2 is much rarer and can cause instability in complexes (Fig. 1)9. Triazoles can also coordinate to metals as N-heterocyclic carbenes rather than through the N donors10.

From our perspective, the coordination chemistry of these triazole ligands holds great promise for the development of metal-based antibiotics11. This area is becoming increasingly relevant due to the rise of antimicrobial resistance (AMR) to the current arsenal of antibiotics, which has been designated by the WHO as one of the top global health threats12,13. Concurrently, there are very few antibiotics being developed, with only 19 antibacterial drugs being approved for use between 2013 and 2023, and of these only 2 represent a new chemical class14. There have been several examples of bidentate pyridyl-triazole ligands being used for therapeutic applications, from using CuAAc-formed triazoles to anchor sulfonamide drugs to metal centers (increasing activity against methicillin-resistant Staphylococcus aureus (MRSA), Fig. 1A)15, to using bidentate triazole ligands directly in forming tris-homoleptic ruthenium [Ru(L)3]2+ complexes (showing broad spectrum antibiotic properties, Fig. 1B)16, tris-homoleptic osmium mer-[Os(L)3]2+ complexes (photoactive against MRSA, Fig. 1C)17 and rhenium(I) carbonyl complexes (displaying activity vs. both Gram-positive and Gram-negative bacteria)18. Additionally, similar bidentate pyridyl-triazoles have been coordinated to a manganese(I) tricarbonyl scaffold, utilizing Clotrimazole as an axial ligand, and were demonstrated to be active against Gram-negative bacteria (Fig. 1D)19. In terms of other therapeutical applications, a ruthenium(II) arene (Fig. 1E) scaffold was coordinated to derivatized sugar pyridyl-triazole ligands (selective activity against ovarian cancer cells)20, and pyridyl-triazole ligands were used to coordinate cyclometallated iridium(III) scaffolds, generating several compounds with photoluminescence properties (Fig. 1F)21. These could be relevant for photodynamic therapy applications against either bacteria or cancer cells, similar to recent work on cyclometallated complexes by Kench et al.22,23. Platinum compounds containing triazole ligands or linkers have also been investigated for anticancer therapy24. Although an impressive array of compounds has been made utilizing triazole ligands25, it should be noted is that in almost all cases, only a handful of individual complexes have been synthesized and tested for their intended applications. One of the reasons for this is the synthesis of the triazole ligand, which requires an azide to be generated as a precursor. Azide formation typically occurs in batch via a reaction between sodium azide and an aryl/alkyl bromide, requiring forcing conditions and undesirable solvents, and can generate significant impurities26,27. Recent developments by Meng et al.28 have opened the door to high-throughput synthesis of this class of ligands through sulfur(VI) fluoride exchange (SuFEx) chemistry under ambient conditions, efficiently generating azides in situ from virtually any primary amine (Fig. 2). We envisaged that this methodology could be used to generate substantial and varied libraries of pyridyl-triazole based ligands. Notably, these ligands offer stability benefits over Schiff-base type counterparts, which are prone to hydrolysis and degradation in the presence of acid or base. Coordination of this library to various metal scaffolds in a similar vein to previous work by our group and others using combinatorial chemistry approaches with Schiff-base ligands29–31 would allow us to create libraries of complexes. These could in turn be screened for antibacterial hits, toxicity towards mammalian cells and other potential areas of discovery such as catalysis and photophysical applications.

To rapidly cover and focus in on the vast chemical space represented by this chemistry, we have turned to a “Direct-to-Biology” screening approach, which has been successfully used across academia and industry22,32–41. This approach involves the high-throughput synthesis and characterization of compound libraries, followed by biological testing of reaction crudes without purification. Concurrently, the reaction components (in our case metal scaffolds and ligands) are tested to support that any observed biological activity is likely from the putative metal complex. There are potential issues with impurities having an outsized effect in these assays, so high yielding reactions, such as those typically used in combinatorial chemistry are favored29–31. As a result, while it is possible to get false positives with a Direct-to-Biology approach, false negatives are rare. The final step is hit validation, where target compounds which showed promising properties in the crude-screening are re-synthesized and purified, and finally re-tested to confirm their biological activity. Overall, Direct-to-Biology represents a powerful screening technique that allows for coverage of a large amount of chemical space quickly, reduces time wasted in making inactive compounds, and funnels down to a small number of compounds with desirable properties. A similar approach can also be taken with photophysical and catalytic testing42,43.

Results
Preparation of pyridine-triazole ligand libraries
We began by optimizing the conditions described by Meng et al.28 for the in situ generation of azides from primary amines, followed by CuAAc Click reactions with 2-alkynepyridines. Initially, significant alcohol impurities were observed, but this was remedied by changing the copper ligand from tris-hydroxypropyltriazolylmethylamine (THPTA) to tris(2-benzimidazolylmethyl)amine (BimH)3. Additionally, we found that the reported phosphate buffer was not required in our system, and low catalyst/ligand loadings of 2.5 mol% were sufficient.

With the optimized conditions in hand, we used an Opentrons liquid handling robot to generate a library of 96 triazole-4-pyridine (Tz-4-P) compounds (24x amines, 4×2-alkynepyridines, Fig. 3A). The success of these couplings was verified by Liquid Chromatography coupled Mass Spectrometry (LC-MS, Fig. 4). A further library of 96 triazole-1-methylpyridine (Tz-1-MP) compounds (12x alkynes, 8×2-picolylamines, Fig. 3B) was prepared using the same method. Under the same conditions, we also attempted to couple 2-aminopyridine with alkynes to generate a Tz-1-P library, but this was unsuccessful.

The average conversion efficiency (as determined by automated LC-MS chromatogram integration at 254 nm)44 was 75 ± 16%, showing that despite the structural diversity of the amines a generally high conversion efficiency was observed (Fig. 4). In terms of trends across the formation of this library, the electron withdrawing ester group on M11 tended to lead to lower conversions (both for the initial click reaction, and for subsequent coordination), although other electron deficient amines (M13, M17) seem to form ligands in high yielding reactions comparable to electron rich systems (M12, M19). As such, it does not appear that the electronics of the amine building blocks have a significant effect on the outcome of the initial CuAAc reaction. The LC-MS traces of ligands containing 7-aminocephalosporanic acid and ampicillin (M15, M16, respectively) were not as clean as the others, and it was found that these ligands and their metal compounds slowly degrade over time in solution (See Supporting Information (SI) Figs. S24, 25). However, it was decided to include them in further testing as they represented more complex structures with β-lactam motifs, and ideally the coordination of these derivatives to metal centers would enhance their biological effects45. On a practical level, it was discovered that several of the target ligands were poorly soluble in DMSO and tended to precipitate or crystallize out of solution. This was especially prevalent among Y4 ligands, where the pyridyl bromine group seem to have significantly affected solubility. This was remedied by pulverizing the affected samples through sonication and aspirating the mixture to homogenize it before dispensing the ligands for further reactions.

Similarly, the Tz-1-MP library was synthesized with a high average conversion efficiency (77 ± 12%), with no noticeable degradation of the products (Fig. 5). Initial tests included two aliphatic alkynes (cyclohexylacetylene and cyclopropylacetylene), but the products of these reactions were not observable by LC-MS. As these alkynes also exhibited problematic volatility, the building blocks were restricted to aromatic alkynes only. These were in turn limited to relatively simple derivatives, which demonstrates a limitation of using aromatic alkynes (Tz-1-MP) compared to primary amines (Tz-4-P) for triazole formation. Conversely, the use of picoline-derived amines allowed for a substantially larger variety in the pyridine fragment of the ligand compared to the Tz-4-P library, with several halogenated derivatives included. Although the structural variety of the Tz-1-MP library is not as broad as Tz-4-P, the overall conversion is comparable, with only A3 (highly electron deficient p-nitro group) and P4 showing consistently lower conversions. No solubility issues were observed with the Tz-1-MP library.

Preparation of metal complex libraries
After test coordination reactions using M4Y1 to optimize conditions, the entire Tz-4-P library was coordinated to the following metal scaffolds (Fig. 3A): [Ir(ppy)2Cl]2 (ppy=2-phenylpyridine, IrCN), [IrCp*Cl2]2 (IrCp*), [Ru(p-cymene)Cl2]2 (RuCy), Re(CO)5Cl (Re(CO)3) and Mn(CO)5Br (Mn(CO)3). These scaffolds were chosen due to their previously demonstrated biological activities and their amenability to combinatorial chemistry19,23,30,31. Both manganese and rhenium scaffolds were pre-activated with silver triflate, and solvent libraries without 1-benzyl-2-methylimidazole (BeMeIm) as the axial ligand were also generated as controls. The reaction mixtures for the five different metal complex libraries were set up simultaneously in a semi-automated high-throughput manner using the Opentrons OT2 liquid handling robot.

Coordination of the Tz-4-P library to the IrCN scaffold was achieved under conditions far less forcing than current literature procedures, which require toxic, high boiling point solvents, temperatures >150 °C, or microwave conditions22,42. In our procedure, high conversion was achieved by heating the ligands and [Ir(ppy)2Cl]2 dimer in a 1:1 mixture of DMSO:H2O at 60 °C for 18 h. IrCp* and RuCy libraries were generated in good purity under ambient conditions. The IrCp* library is being explored in an ongoing project for biological activity, so will not be discussed further here.

For the Re(CO)3 library, poor conversion to the target complexes was initially obtained (1:2 MeOH/DMSO, 70 °C, 18 h). After addition of an excess of MeOH, the reaction crudes were left standing for one week at room temperature to allow for the solvent to evaporate, giving significantly improved product formation. This is likely due to DMSO solvent competitively binding with the rhenium scaffold instead of BeMeIm. A similar effect was observed for the Mn(CO)3 library, as when conversion is low, the byproduct is often the solvent adduct (e.g. Mn(CO)3 M2Y3, see Supporting Information Figs. S26, 27). Previously, our group has shown that manganese(I) carbonyl complexes with solvent as the axial ligand tend to have low antibacterial activity31. Manganese(I) tricarbonyl complexes are often light-sensitive, so this library was prepared, handled, tested and stored in the dark.

Test coordination to the same scaffolds using Tz-1-MP (P1A1) implied that DMSO bound competitively with all metal centers, preventing coordination in many situations, even at elevated temperatures. The exception to this was the [IrCp*Cl2]2 scaffold, which yielded product and led to the creation of an IrCp* library (Fig. 3B). In addition, excess EtOH with [Ru(p-cymene)Cl2]2 gave the desired complexation with P1A1 in high yields, so the corresponding RuCy library was prepared with the Tz-1-MP ligands (Fig. 3B).

The poor complexation of the Tz-1-MP library could be due to the greater flexibility imparted by the methyl linker, forming 6-membered metallocycles rather than 5-membered metallocycles formed by the more rigid Tz-4-P library. Generally, rigid 5-membered rings are significantly more stable than 6-membered rings due to fewer degrees of freedom and a more optimum “bite angle” (the angle between the donor groups and the metal center)46. More specific examples involving Tz-1-MP scaffolds from Urankar et al. show that under certain coordination conditions, these ligands can bind in a monodentate fashion through the triazole ring (to palladium) rather than a chelating effect47. Maisonial et al. similarly demonstrated that for triazoles with a pendant primary amine, 5-membered chelates were significantly more stable when coordinated to platinum than 6-membered metallocycles48.

Typically for metal-based combinatorial synthesis, libraries are only partially characterized (either random representative samples or predetermined subsets, e.g. using 19F NMR for fluorine containing compounds) to reduce the time-consuming characterization step to a more practical level29,42,43. In our case, the charged nature of the metal complexes makes them amenable to characterization by LC-MS, a technique that itself can be highly automated both in collection and assessment of the data (see Supporting Information Section 2.9). Therefore, the purity and identity of all compounds were assessed and the processed LC-MS spectra, target complex percentage conversion and retention times are supplied as Supplementary Data 1 with this article.

For Mn(CO)3, and to a lesser extent the Re(CO)3 library, Y2 containing ligands were consistently poor at coordination. This could be due to steric clashing of the pyridine methyl group with the axial ligands during coordination. Similarly, Y4 containing ligands performed poorly, possibly due to the lower solubility of the ligands, or electron withdrawing effect of the pyridyl para-bromine. Generally, ligands formed from simple aryl amines such as M8, M10 and M17 tended to coordinate most efficiently. Our data also indicates that M group sterics do not affect the yields, likely due to these groups being spatially removed from the metal center. Electronic effects seem to be more consequential, with strong electron donating units such as methoxy- and methyl-groups favored. Aliphatic groups from building blocks such as M1, M2 and M3 also coordinated well, again implying the prevalence of electronic effects on coordination over steric influences.

For the Tz-1-MP library, coordination of the P6 variants was consistently poor, likely due to the gem-dimethyl groups on the methyl linker. As well as being a useful functionality in medicinal chemistry49, it was envisaged that the Thorpe-Ingold effect would be beneficial to coordination, both increasing rotational barriers and decreasing the bite angle of the ligand50. Instead, it appears that the steric bulk of the gem-dimethyl groups effectively inhibits chelation in P6 containing ligands. In most other cases, coordination of this library to the metal centers was reasonably successful, and the general high yielding formation of this ligand library by CuAAc reaction is a testament to the robustness of the optimized Click conditions.

Photophysical evaluation and catalytic testing
Taking inspiration from the work by Kench et al., we investigated the photophysical properties of the crude IrCN library with the aim of exploring the possibility of photodynamic therapy applications22. A range of fluorescence intensities was observed, but the maximum emission wavelength (λmax) was evidently dependent on the Y alkyne substituent (see Supporting Information, Fig. S18). This indicates that chemical modifications on the pyridine (Y) are generally more electronically impactful than the amine modifications (M, affecting the triazole). Information about the excited state identity and lifetime can be inferred from the shape of the emission spectra42. The typical excitation and decay cycle of cyclometallated iridium(III) complexes involves excitation from the ground state to a singlet metal/ligand-ligand charge transfer excited state (MLLCT, dπ→π* transition), followed by intersystem crossing to a triplet 3MLLCT excited state. The decay of this pure excited state is often rapid and is a single peak with a roughly Gaussian distribution, such as observed in Y2 and Y4 complexes. Mixing of the 3MLLCT state with a ligand-centered triplet state (3LC) can also occur, giving a longer-lived excited state. This is characterized by a peak with a significant shoulder (Y1 and Y3). The longevity of the excited state can have significant impacts on the photophysical properties of molecules, including on the generation of reactive oxygen species (ROS).

To ascertain whether ROS was generated by the IrCN library, irradiation at 405 nm with ROS scavengers (ABDA (anthracenediyl-bis(methylene)dimalonic acid) and RNO/histidine (p-nitrosodimethylaniline)) was undertaken. ABDA was observed to degrade in the DMSO:PBS media, a previously reported effect upon irradiation below 450 nm51. Significantly less ROS was detected for all compounds compared to the Ru(bpy)3 2+ (bpy=2,2’-bipyridyl) control using the RNO/histidine assay52. This suggests that ROS generation for these IrCN compounds is occurring at a lower rate. Altering the cyclometallated ligand may result in complexes with higher ROS-production rates.

Following combinatorial work on ruthenium arene Schiff-base complexes by Weng et al., we tested the crude RuCy libraries for transfer hydrogenation (TH) catalysis using the Coumarin-N3 assay developed by the same group43. As Coumarin-N3 is hydrogenated to the amine, it becomes fluorescent, so the formation of the product can be monitored over time (Fig. 6A). The rationale behind this reaction is to take advantage of high formate concentrations in E. coli and S. aureus to hydrogenate azide pro-drugs in bacteria53. As can be seen in Fig. 6B, C, which shows the change in fluorescence (Δ fl) intensity from TH catalyzed by the RuCy libraries, RuCy(Tz-4-P) type compounds (Fig. 6C) are generally more active than the RuCy(Tz-1-MP) library (Fig. 6B), with 3 compounds (M11Y2, M13Y2, M24Y2) all having a Δ fl > 15000 arb. units, comparable with active compounds reported by the Ang group. Interestingly, the most active compounds all contain the Y2 motif with an electron donating methyl group in the 2-position, which seem crucial for activity.

We additionally tested the crude IrCp* libraries for transfer hydrogenation in an assay adapted from Miller et al. for evaluating the activity of iridium-based artificial metalloenzymes using harmaline (Fig. 6D–F)54. Iridium-based TH systems tend to be the most active compared to other transition metal catalysts55, and for the harmaline assay this was demonstrated to be the case. Preliminary testing with the RuCy(Tz-1-MP) library showed no activity, while several of the IrCp*(Tz-4-P) (Fig. 6F) complexes showed high conversions of the harmaline starting material to product (measured by a change in absorbance (Δ abs) at 384 nm corresponding to harmaline consumption, confirmed by LC-MS, Figs. S10, 11). In the cases where good conversions were observed, the electron donating methyl and methoxy groups (Y2, M14, M23) as well as bromine groups were present (Y4). This is in line with previous work in the field where small changes in ligands can have dramatic effects on the activity of the catalyst56. This can be ascribed to a complicated interplay of steps in TH, which first require abstraction of the coordinating chloride, formation of a hydride species (favored by electron rich ligands)57, substrate coordination (aided by labile ligands, often electron poor)58 and concurrent transfer of hydride (enhanced by electron rich ligands). Upon testing the IrCp*(Tz-1-MP) library (Fig. 6E), it became apparent that these catalysts were significantly more active than their Tz-4-P counterparts. As 6-membered chelates, they are more labile while remaining electron donating, which could explain the higher catalytic activity47,48. Additionally, the IrCp*(Tz-1-MP) library tended to have a greater proportion of MeCN adduct (M2+) peaks in the LC-MS traces than the IrCp*(Tz-4-P) library, indicating that halide abstraction is easier.

As such, we decided to re-synthesize and re-test two of the most active complexes, IrCp*(P1A7) and IrCp*(P4A11). The lability of 6-membered chelates meant that for both compounds, some free ligand was observed in solution by NMR spectroscopy even after purification. In the harmaline assay, only IrCp*(P4A11) demonstrated activity, on par with its crude. Upon examination of the crude LCMS of IrCp*(P1A7), there were a couple of significant impurities including free ligand and an unknown iridium(III) dimer, suggesting that the parent complex was not the catalytically relevant species. In contrast, the crude LCMS of IrCp*(P4A11) only showed the parent complex and the [solvato-iridium]2+ species (See Supporting Information Section 2.6 for LC-MS traces and kinetic graphs). Interestingly, both purified catalysts were active in the coumarin-N3 assay, with IrCp*(P4A11) displaying superior activity. Both catalysts surpassed the activity of the best crude RuCy catalysts (Table 1, Δ fl=46056, 31295, 16505 arb. units for IrCp*(P4A11), IrCp*(P1A7) and RuCy(M13Y2), respectively)

We further investigated the effect of exogenous chloride by testing the two pure catalysts in PBS 1X/H2O co-solvents with tBuOH, with and without halide extraction by AgOTf (Fig. 6G–I). As can be seen in Table 1, removing excess chloride accelerates the reaction in all cases, likely due to biasing the equilibrium towards the complex required for catalysis58. For IrCp*(P1A7), pre-activation with AgOTf did indeed accelerate the reaction (1.4x faster), even in the presence of exogenous chloride (1.1x faster) in the harmaline assay, suggesting that initial chloride abstraction is the key energetic barrier for this catalyst. On the contrary, in the same assay, IrCp*(P4A11) did not benefit from pre-activation in both water (no change) and PBS (1.4x slower), suggesting that a different part of the mechanistic cycle is the turnover-limiting step. We envisage that these catalysts could be applied to molecular uncaging in bacteria or even in mammalian cells43,56,59, but further mechanistic analysis and assay development would be required, which is beyond the scope of this work.

Overall, we have demonstrated that these metal complex libraries can be rapidly assessed for photophysical and catalytic properties using 96 well plate-based assays under ambient conditions. We envisage that any property-screening assay that is amenable to a well plate format could be adapted to test these and other combinatorial metal complex libraries to identify ideal compounds. We are currently exploring further optimization of metal scaffolds in this area.

Biological testing of synthesized libraries
With the main focus of our group being the exploration of transition metal complexes as antimicrobial agents, we advanced all the prepared libraries to an initial biological assessment, using a “Direct-to-Biology” approach. In a primary screening, all crude reaction mixtures were evaluated against two bacterial strains, one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). As hit-rates against Gram-negative strains are generally much lower60, we decided to evaluate compounds against E. coli at 50 µM and against S. aureus at 12.5 µM and 50 µM to be able to better differentiate the level of activity. Metal-based compounds have a reputation of being generally toxic, although recent reports have questioned this assumption, at least in the realm of in vitro assays11. We therefore tested compounds against human embryonic kidney cells (HEK293T) at 50 µM. Amongst the 192 Tz-4-P and Tz-1-MP ligands, only one reduced the average viability of HEK293T cells below 50% (M12Y3, 45±7% cell viability), showing that the ligands on their own are generally non-toxic (Fig. 7A, B). None of the ligands showed any significant activity against E. coli, and only four showed growth inhibition against S. aureus: M7Y1 (50 µM), M16Y2 (12.5 µM), M16Y3 (12.5 µM), and M23Y3 (50 µM). As M16 contains the broad-spectrum antibiotic ampicillin and the reactions to form those compounds had relatively lower conversion, the observation of some antibacterial activity is unsurprising. We conducted dose-dependent testing of these crudes against S. aureus to determine minimum inhibitory concentrations (MIC) of the putative ligands and found them to be 50 µM for M7Y1 and M23Y3, while lower MICs of 12.5 µM and 3.13 µM were observed for the ampicillin containing triazole compounds. Preliminary testing on the metal complex precursors ([Ir(ppy)2Cl]2, [Ru(p-cymene)Cl2]2, Re(CO)5Cl, Mn(CO)5Br) showed that only [Ir(ppy)2Cl]2 had an MIC of 50 µM against S. aureus., with the rest having a values > 50 µM. Overall, almost all the ligands showed no significant antibacterial or cytotoxic properties, and alongside the lack of activity of the metal scaffolds, this allows us to assign any biological effects observed on the metal complex crudes to the putative metal complex with high confidence.

All but one of the IrCN complexes showed antibacterial activity against S. aureus (Fig. 7C). However, none showed any appreciable effect on the growth of E. coli. This finding aligns with the general difficulty across the field of (metallo)antibiotic drug discovery to find compounds able to effectively penetrate the double-membrane and evade the efflux pumps of these pathogens61,62. Dose-dependent screening of the IrCN libraries against S. aureus revealed high antibacterial activity. The lowest MIC measured was 0.39 µM for four compounds which is comparable to the standard-of-care antibiotic vancomycin (MIC=0.42 µM), which was used as a control. Overall, 59 out of 96 compounds displayed an MIC < 5 µM.

A crucial parameter of any antibiotic is its selectivity for bacterial cells over healthy human cells, often denoted by the Therapeutic Index (TI). Unfortunately, most of the IrCN also significantly reduced human cell viability at 50 µM. However, given the very high level of antibacterial activity and the observation that the inhibition of a significant portion of the compounds was in the 25–50% range, we identified several compounds with a potentially viable therapeutic index of >10.

The Re(CO)3 compounds similarly showed high levels of antibacterial activity in S. aureus, with 61 compounds completely inhibiting bacterial growth at 50 µM or lower (Fig. 7D). The MICs were generally higher than for the IrCN library, but the compounds were consistently less toxic. Most caused less than 50% growth inhibition in the HEK293T cells, while still having MICs as low as 0.78 µM.

Comparing the Re(CO)3 and IrCN libraries, there is a clear mirroring of activity for individual ligands. Generally, if a Re(CO)3 complex with a given ligand is active, then the corresponding IrCN will be equally or more active. This is noteworthy as the ligands themselves are demonstrably not active and the two metal scaffolds are clearly distinct. We speculate that the arrangement of the ligand in an octahedral geometry and the occupation of the coordinating nitrogen atoms with the metal scaffold may be the cause for this.

For the Re(CO)3 library, there is a correlation between its activity and conversion efficiency. The same was not observed for the IrCN library. In terms of specific trends, Y2 and Y4-derived ligands are much less active for Re(CO)3 than IrCN, which can be ascribed to lower conversion efficiency in the former. Y1 and Y3-derived ligands are the most active for Re(CO)3, with active compounds having similar activity in both metal libraries. Indeed, M17–22 for Y3 shows almost identical activity for both Re(CO)3 and IrCN, which suggests that the identity of the metal scaffold is not necessarily important for activity. M5 (enantiopure (S)-1-phenyl-ethylamine)-derived ligands are the overall most active for Re(CO)3, and are similarly highly active for IrCN, while there was no or very low activity for: M2 (pendant amide, despite high conversions), M11 (phenyl-ester, which showed good activity for IrCN despite low conversions), M15 (β-lactam, poor conversions), and M23 (biaryl, also poor conversions).

By contrast, the Mn(CO)3 compounds generally showed lower to no significant antibacterial properties than its congener library (Fig. 7E). Only 21 compounds showed any antibacterial activity against S. aureus and only 4 had an MIC < 10 µM. Nevertheless, the overall hit-rate of 21% is comparable to the hit-rate we previously observed in Mn(CO)3 Schiff-base libraries of 15.3%31. The hit-rate is also still much higher compared to what is generally found in purely organic molecule screening campaigns (~1%)62. At the same time, this compound class showed similar rates of cytotoxicity to rhenium, with half of all compounds causing cellular growth inhibition of less than 50% at 50 µM, and a handful causing greater than 75% growth inhibition.

Lastly, the RuCy libraries with both Tz-4-P and Tz-1-MP ligands (Fig. 7F, G) showed the lowest level of antibacterial properties. Only a few of the RuTz-4-P compounds inhibited growth against S. aureus at 12.5 µM. This is in slight contrast to previous work on ruthenium p-cymene Schiff-base compounds, which have shown high hit-rates and significant antibacterial activity30,38,43,63.

Structure-Activity Relationship (SAR, see Supporting Information Section 4) analysis was performed on the data gathered to gain insights into what features contribute most to activity and toxicity. A strong correlation between the retention time (RT) of the Tz-4-P library by LC-MS and calculated cLogP values for the ligands indicates that RT is a good descriptor for lipophilicity in these libraries. For the Re(CO)3 library, the active compounds tended to have a tighter range of RTs than non-active compounds, and the molecular weight (MW) was also important (low MW compounds tending to be more active). This is in line with guidance for organic antibiotics, which indicates that lighter and more polar molecules have better uptake in bacteria64. Conversely, a weak trend between MW and toxicity suggests that heavier compounds are less toxic. An analysis of the fraction of carbons sp3 character suggested that there is an optimum window that imparts high activity, which can be rationalized by the ligand structure affecting uptake into bacteria. There was also a clear correlation between the antimicrobial activity and toxicity of this library, with more active compounds being more toxic. However, at MIC 6.25 µM, most compounds are in the 50% viability range for HEK293T cells at 50 µM, so there is a good potential therapeutic window.

For the IrCN library, the trends were not as clear, but there was a weak correlation between MW and activity, with more active compounds being lighter, as well as between MW and toxicity. There is also a weak positive correlation between activity and toxicity. For a given ligand, the IrCN complex tended to be significantly more active than the same Re(CO)3 compound, which was in turn more active than the Mn(CO)3 complex. This suggests that for a given ligand, the identity of the metal scaffold has a larger effect on the biological activity of the resultant compound than the identity of the ligand.

Some further insights into more concrete structural contributors to antimicrobial activity in metal complexes were obtained by training a support vector machine machine learning (SVM-ML) model on the antibacterial activity data. The structures of rhenium and iridium metal complexes (which had the most interesting levels of antibacterial activity) were converted to a 598-bit ELECTRUM fingerprint based on our recent work65. Due to the limited dataset size of 192 compounds, we converted the antibacterial activity data into binary classification data (a compound was labelled active (“1”), if MIC value was ≤ 6.25 µM or inactive (“0”) otherwise), which is commonly done even for larger datasets of antimicrobial compounds66. The SVM-ML model was trained with 5-fold cross validation, and the trained model showed strong performance in predicting compound bioactivity. It achieved a mean AUC of 0.83±0.05, indicating good ability to discriminate between active and inactive compounds. The mean precision was 0.80±0.09, demonstrating that roughly 80% of compounds predicted to be active were truly active. Similarly, the mean recall was 0.87±0.06, showing that the model successfully identified the majority of active compounds. Overall, the balance between precision and recall is reflected in a mean F1 score of 0.83±0.07, suggesting reliable and consistent hit prediction across cross-validation folds. This suggests that detailed molecular structural features within the compounds correlate with their biological activity. We further analyzed the feature importance through the support vectors of the SVM. Structural features that correlated to antibacterial activity against S. aureus included: amide bonds, fluorine substituents and higher substitution patterns around the metal coordination site (Fig. S46). Conversely, ether groups and alkyl amines correlated with inactive compounds (Fig. S47)

In summary, we have efficiently evaluated 192 ligands and 480 metal complexes for their antibacterial and cytotoxic properties (an additional 768 IrCp compounds are currently undergoing evaluation in ongoing work). This “Direct-to-Biology” methodology allows us to rapidly narrow our focus on potentially interesting compounds with a desirable therapeutic index, and the combination of single-dose response and full MIC assays on crude reaction mixtures provides key insights into activity trends across metal libraries.

Re-synthesis and biological testing for lead compounds
Based on the MIC and single dose response toxicity data of the crude reaction mixtures, 6 complexes were chosen for re-synthesis, full characterization and re-testing (2x Re(CO)3, 2x IrCN and 2x Mn(CO)3). The criteria for choosing these was based primarily on antimicrobial activity, with the most active complexes identified (no compounds with an MIC higher than 6.25 µM were considered). Of these, the least toxic compounds were selected. For the IrCN library, as almost all compounds caused less than 50% cell viability, the least toxic compounds with MIC of 1.56 µM were chosen. The Re(CO)3 appeared to provide a good balance of active compounds with low apparent toxicity, so again the most active and least toxic compounds were chosen. For the Mn(CO)3 library, only four compounds had an MIC of 6.25 µM, so the purest compounds by LC-MS were selected for re-synthesis.

The synthesis of all compounds was achieved by first making the ligands using the scaled-up procedure for generating ligand libraries, changing the solvent from DMSO to DMF to enable a more feasible workup and purification. The ligands were then reacted with the metal in high-yielding reactions. After purification by flash column chromatography, the pure compounds were fully characterized. Single crystals of Mn(CO)3 (M19Y1), Re(CO)3 (M1Y1), and Re(CO)3 (M20Y3) were obtained by slow vapor diffusion (CHCl3/Et2O, DCM/n-hexane, and DCM/Et2O respectively), with the X-ray diffraction structures (Fig. 8) clearly showing the bidentate binding mode of the pyridine-triazole ligands.

Stability assays on the pure compounds were undertaken, with biologically relevant solvents and mixtures tested (DMSO, H2O, 1X PBS and a combination of DMSO with H2O and 1X PBS). The stability was ascertained by measurement of UV absorbance (320 nm) at 37 °C over 21 h. Most compounds demonstrated good overall stability with less than 10% difference in the spectra over this period; however, the Mn(CO)3 complexes exhibited significant changes in their overall UV-Vis spectra in H2O and 1X PBS over 2–8 h. This could be due to anion exchange (in the case of PBS) or water competitively binding with the complex in the axial position. However, this did not seem to be detrimental to the antibacterial activity of these compounds (vide infra).

Biological studies were undertaken on the purified compounds to confirm activity against bacteria (MIC assays) and ascertain toxicity (CC50 measurements against HEK293T cells). Almost no activity was observed against E. coli at the measured concentrations, with the exception of Re(CO)3 (M1Y1) and Mn(CO)3 (M22Y1) (MIC=100 µM, see Supporting Information Table S5). However, perturbation of the outer membrane by polymyxin B nonapeptide (PMBN) resulted in significant activity against E. coli for all compounds (see Supporting Information Table S6)67. PMBN has no inherent antibacterial activity, but damages and weakens the outer membrane of Gram-negative bacteria by interacting with the anionic lipopolysaccharide molecules. This permeabilizes the membrane to antibiotics, suggesting that uptake into Gram-negative bacteria is the issue for these compounds and they are not necessarily Gram-positive specific68.

For the Gram-positive strains, high activity was observed in almost all cases. A comparison of activity between the crude reaction mixtures and re-synthesized compounds against S. aureus gives an increase in activity in all cases, with very low MICs (5/6 compounds active in the nanomolar range, similar to the vancomycin control MIC=0.42 µM, Table 2). This confirms that in all cases it is the putative metal complex in the crudes that is responsible for the observed activity, validating our methodology of pre-screening large crude libraries (Table 2). It should be noted that in several cases (Re(CO)3(M1Y1), IrCN(M12Y1), Mn(CO)3(M19Y1)) there were larger increases (>16x, >4x, and >8x more active respectively) in antibacterial activity than expected from the increase in purity due to re-synthesis (crude purity by LC-MS of 43%, 72%, 41% respectively). This suggests that our methodology in fact underestimates the activity of the complexes in the synthesized libraries.

The other tested Gram-positive strains showed generally good activity, with slightly higher MICs against Enterococcus faecalis in most cases. The exception was the manganese complexes, which were significantly less active. Enterococcus faecium showed very low growth in the Mueller-Hinton (MH) growth medium that was typically used, so Tryptic Soy Broth (TSB) growth medium was used instead. This gave measurable MICs, but a comparison with E. faecalis (which was tested in both MH and TSB) indicates that TSB results in a significant rise in MIC values. As such, although the MICs for E. faecium are higher than S. aureus, there is still good activity in most cases (Table 2). Generally, Re(CO)3 (M20Y3) and IrCN(M12Y1) show exceptional activity across the tested Gram-positive (and sensitized Gram-negative) bacterial strains, with often nanomolar MICs. The fact that the compounds showed very low MICs against E. coli upon permeabilization suggests that further structural optimization targeted at improving Gram-negative bacterial uptake could result in pan-active metalloantibiotics. As the antibacterial activity of the pure compounds increased compared to the crude, the same trend was also observed for their cytotoxicity. Indeed, most of the compounds displayed CC50 in the range of 10 µM. However, due to their high level of antibacterial activity, all compounds except Mn(CO)3 (M22Y1) still displayed a favorable therapeutic index >10. The best ratio of antibacterial activity and toxicity was determined for IrCN(M8Y4) showcasing a therapeutic index between 49–99. Overall, these values are comparable and in several cases superior to that of other recently reported metal-based antibiotics16,23,31.

Additional toxicity testing in the form of haemolysis was undertaken. For human red blood cells, the concentration at which 10% of them have lysed (HC10) was low to moderate for the two Re complexes (HC10 = 36±6 µM and 73±29 µM, respectively), mirroring their toxicity against mammalian cells. This was also true for IrCN(M12Y1) and Mn(CO)3 (M22Y1), which also caused haemolysis, albeit at higher concentrations (HC10 = 81±4 µM and 87±7 µM respectively). Interestingly, both IrCN(M8Y4) and Mn(CO)3 (M19Y1) showed no haemolysis at any of the tested concentrations (HC10 > 200 µM). For IrCN(M8Y4), this is especially encouraging, as it is highly active against Gram-positive strains, has the highest CC50 tested and is not haemolytic. For all compounds, the HC10 values are significantly higher than the MIC concentrations against Gram-positive strains, so there is still a potential therapeutic window available.

From a methodology perspective, the single dose toxicity assay was used as a guide to select the most active and least toxic compounds for re-synthesis. It is clear from the CC50 data that although the single dose assay mirrors the true toxicity, it can underestimate the cytotoxicity of the pure compounds. For the IrCN compounds, it was expected that they would have significant toxicity based on recent work, and the single dose assay did indeed indicate that 22. More surprising is the Re(CO)3 data, which was significantly more toxic than expected from the preliminary assay. As such, the high throughput assay does successfully indicate toxicity, but more stringent conditions on selecting active and non-toxic compounds will be used in future work, such as increasing the toxicity threshold above 50% cell viability. It should be noted that cell-based assays can only provide so much information and hit compounds will have to undergo more stringent testing before a conclusion can be made about their safety.

In summary, we have presented a combinatorial methodology to generate large libraries of pyridyl-triazole bidentate ligands formed by CuAAc Click reactions. These libraries have been demonstrated to coordinate efficiently to a number of metal scaffolds, generating 672 well-characterized complexes under mild conditions. We have showcased some applications of our metal libraries for catalytic and photophysical applications, including synthesizing and testing two iridium complexes in transfer hydrogenation assays. We have successfully screened all compounds for antimicrobial activity and toxicity in a high-throughput “Direct-to-Biology” approach, and, using the available data, have re-synthesized several highly active metalloantibiotics, with MICs as low as 200 nM against S. aureus. We have also demonstrated that the single-dose antibacterial and toxicity assays are a reliable indicator of the properties of the pure compounds from crude reaction mixtures, even for low-yielding reactions. However, care must be taken when it comes to single-dose toxicity assays as they tended to indicate that compounds are less toxic than they turned out to be upon re-synthesis.

We have identified one complex by our Direct to Biology approach, IrCN(M8Y4), which has good activity against Gram-positive strains and relatively low toxicity against HEK293T cells, giving it a favorable therapeutic index (>49 against S. aureus). Additionally, it has been shown to be non-haemolytic and demonstrates good stability in a variety of relevant solvents. These properties make it a potential candidate as an antibiotic against systemic infections. Further testing will involve advanced toxicity and stability studies, efficacy against a broader panel of Gram-positive bacteria, and potential in vivo testing.

Although promising activity against Gram-positive bacteria has been reported here for a number of different metal complexes, activity against Gram-negative strains has been lacking. Improved activity in the presence of the PMBN sensitizer suggests that the low original activity is in part due to poor uptake of the synthesized metal complexes. However, thanks to the combinatorial and versatile ligand architecture there is now scope to significantly vary the ligands to explore and include motifs to improve uptake into bacteria69,70.

Screening crude reaction libraries for catalytic applications has also been demonstrated, with 384 complexes evaluated across two different transfer hydrogenation assays. These assays are amenable to high-throughput reaction monitoring under ambient conditions in 96-well plates and allowed us to identify and resynthesize two promising IrCp* catalysts. One of them, IrCp*(P4A11), was highly active in both assays and shows promise for further catalytic testing, including prodrug unmasking in cells. Work in our group is currently ongoing with derivatives of IrCp* and IrCN libraries to explore photophysical properties, photodynamic therapy, and catalytic applications.

The potential scope of this ligand chemistry is somewhat limited by the availability of 2-alkynepyridines (for the Tz-4-P library). However, this limitation is more than made up for by the capacity to use practically any primary amine to generate bidentate ligands, and hence a large number of metal complexes are now obtainable. Additionally, as the ligand architecture is highly variable around a fixed core motif, this methodology lends itself well to potential machine learning applications to enable the discovery of highly optimized compounds30. In terms of other biological applications, assays for any disease-based screening would in principle be possible on these libraries or derivatives of them.

We believe there is much scope to expand the types of chemistry that are amenable to the combinatorial generation of ligand structures described here. Additionally, we have only scratched the surface of the periodic table in terms of explored metal scaffolds. While each metal may require slightly different conditions and not all of them will be amenable to this type of chemistry, we believe that the field of combinatorial metal complex synthesis is only just at its beginning.

Methods
Generalequipmentandsoftware
NMRspectrawereobtainedusingaBrukerAvanceNeo700instrumentfittedwithaliquidN2cooledtripleresonancecryoprobe(700MHz[1H]),BrukerAVIIIHD600Wideboreinstrument(600MHz[1H]),orJEOLECX400orJEOLECS400spectrometer(400MHz[1H]).High-resolutionmassspectrometry(HRMS)ESI-MSspectraweremeasuredusingaBrukerDaltonicsmicrOTOFMS,Agilentseries1200LCwithelectrosprayionization(ESI)oronaThermoLCQusingelectrosprayionization,with<5ppmerrorrecordedforallHRMSsamples.LCMSdatawascollectedusingaDr.MaischReproSil-Pur120ODS-3,2.4µm,50mm×3mmcolumn,onaThermoScientificVanquishinstrument,coupledtoaThermoScientificHCTultraETDIIiontrap.ThesolventsystemconsistedofH2O/MeCN,bothcontaining0.1%formicacid.Characterizationdataandassessmentsofpurityonthegeneratedlibrariesweredoneusing254nmabsorbances.UV-VisabsorbanceandfluorescencedatawerecollectedusingaTecanInfinite200Proplatereader,usingthe“Greiner96FlatBottomTransparentPolystyrene”option.96-wellreactionswereshakenandheatedusinganEppendorfThermoMixerwith“plates”attachment.Diffractiondatawerecollectedat110KonanOxfordDiffractionXtaLABSynergyHyPix-Arc100diffractometerwithCu-Kαradiation(λ=1.54184Å).ThecrystalswerecooledwithanOxfordInstrumentsCryostream1000.

NMRdatawerecollectedusingBrukerTopspin3.5pl6(600MHz),TopSpin4.1pl3(700MHz).LCMSdatawascollectedusingThermoScientificXcalibur4.4.High-resolutionmassspectrometrydatawerecollectedusingBrukerCompassHyStar4.1.FT-IRdatacollectedusingPerkinElmerSpectrumv10.5.4.UV-Visdata,fluorescenceandbiologicaldatawerecollectedusingTechani-control2.0.XRDdatawerecollectedusingRigakuCrysAlisProv44.128a.DataanalysiswasperformedforHigh-resolutionmassspectrometryusingBrukerCompassDataAnalysis.NMRdatawereprocessedusingMestReNova15.1.0-37919.XRDdatawereprocessedinOlex2-1.5-ac6-020.Biologicaldata(CC50,Haemolysis)wereanalysedandprocessedusingGraphPadPrismv8.0.2(GraphPadSoftware,California,USA).CodewritingwasdoneonVisualStudioCode1.105.1,usingPython3.11.9.

Syntheticmethods
Preparationofazidetransferreagent.MethodadaptedfromMengetal.28NaN3(99mg,1.524mmol,1equiv.)wasdissolvedindeionizedwater(3.0mL)ina15mLFalcontube.Tothis,3.0mLofMTBEwasaddedandthemixturestirredvigorouslyat0°C.Separately,1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-iumtrifluoromethanesulfonate(600mg,1.83mmol,1.2equiv.)wasdissolvedin150µLofMeCNinaFalcontube,andwasaddedtothereactionmixture.Afurther150µLofMeCNwasusedtowashtheFalcontubeandensureefficienttransferofmaterial,withthisalsobeingaddedtothereactionmixture.Themixturewasstirredvigorouslyat0°Cwiththelidlooselyscrewedontopfor15min,thenallowedtosettleatr.t.for30min.Theupperorganiclayerwasdecantedanddilutedwith3mLDMSO,givingasolutionofFSO2N3of~173mM.

Generalprocedureforsynthesisofligandlibraries.MethodadaptedfromMengetal.28.Thefollowingprocedureassumesaconcentrationof173mMforFSO2N3anddescribestheadditionto1wellofaParadox®96wellplate(700µLglassvial)usinganOpentronsOT2liquidhandlingrobot(P300Gen2300µLpipetteandP20Gen220µLpipette).Tothereactionwell,sat.KHCO3solution(11µL,4equiv.,nominally3M),followedbyDMSO(65µL),wasadded,thenaminestocksolution(100µL,80mM,1equiv.).FSO2N3stocksolutionwasthenadded(48µL,173mM1.05equiv.,Note:thepipettewasaspirated3timestoequilibratesolventvaporpressure).Thereactionvialswerethensealed,andshakenat800rpmfor18hatr.t.Sodiumascorbatesolution(80µL,50mM,0.5equiv.)wasaddedandthereactionshakenforafurther15minatr.t.Toeachreactionwell,alkynestocksolution(100µL,80mM,1equiv.)wasadded,followedby(BimH)3solutioninDMSO(10µL,20mM,2.5mol%),thenCuSO4solutioninwater(10µL,20mM,2.5mol%).Thereactionwassealedandshakenat800rpmfor18hatr.t.ThereactionmixturewasanalyzedbyLCMS(5µLin200µLMeCN/H2O(60:40))toconfirmthepresenceandidentityoftheproductligand.

SynthesisofIrCN(Tz-4-P)library.IrCNdimer([Ir(ppy)2Cl]2,34.3mg,0.032mmol)wasdissolvedinDMSO(3.2mL,10mM).Ligandstocksolution(Tz-4-Plibrary,30µL,20mM)wasdispensedinto700µLglassvials,with30µLofIrCNdimerstocksolutionaddedtoeachwell,followedby60µLofwater(giving120µLof5mMcomplexes).Theplatewassealedandheatedat60°C(600rpm)for18h.

SynthesisofRe(CO)3(Tz-4-P)library.Re(CO)5Cl(50.6mg,0.14mmol)andAgOTf(36mg,0.14mmol.Note:hydroscopic,light-sensitive)wereplacedina20mLmicrowavevial.MeOH(7mL)wasadded,andthemixturewasheatedinaBiotageInitiatormicrowavereactorat120°Cfor30mins.Aftercooling,thesupernatantwasusedasastocksolutionof[Re(CO)3(MeOH)3][OTf](20mM).Ligandstocksolution(30µL,20mM)and1-methylbenzimidazolestocksolution(30µL,20mM,DMSO)weredispensedinto700µLglassvials,with30µLofRe(CO)3(MeOH)3OTfstocksolutionaddedtoeachwell.Theplatewassealedandheatedat70°C(600rpm)for18h.Afterthis,240µLofMeOHwasaddedtoeachwell,andtheplatewasleftunsealedforoneweek,allowingfortheMeOHtoevaporateoff(giving60µLof10mMcomplexes).

SynthesisofMn(CO)3(Tz-4-P)library.(Note:Mncomplexesarelight-sensitive,soallstepsweredoneindarkness.Thepre-activationofMnwasperformedunderN2,butotherstepsweredoneunderambientconditions).Mn(CO)5Br(38.5mg,0.14mmol)andAgOTf(36mg,0.14mmol.Note:hydroscopic,light-sensitive)wereplacedina50mL2-neckedroundbottomedflaskfittedwithacondenser,andevacuated/backfilledwithN2threetimes.DryTHF(7mL)wasadded,andthemixturewasrefluxedunderN2at70°Cfor45min.Aftercooling,thesupernatantwasusedasastocksolutionofMn(CO)3(THF)3OTf(20mM).Ligandstocksolution(30µL,20mM)wasdispensedinto700µLglassvials,with30µLofMn(CO)3(THF)3OTfstocksolutionaddedtoeachwell.After2hatr.t.,1-methylbenzimidazolestocksolution(30µL,20mM,DMSO)wasadded,andtheplatewasleftshakingatr.t.(600rpm)for72h,allowingfortheTHFtoevaporateoff(giving60µLof10mMcomplexes).

SynthesisofIrCp*(Tz-4-P)andIrCp*(Tz-1-MP)libraries.di-μ-chloro-bis[chloro(pentamethylcyclopentadienyl)iridium(III)](49.4mg,0.062mmol)wasdissolvedinDMSO(6.2mL,10mM).Ligandstocksolution(30µL,20mM)wasdispensedintoapolystyrene96wellplate,with30µLofIrCp*stocksolutionaddedtoeachwell(giving60µLof10mMcomplexes).Theplatewasleftstationaryfor24hatr.t.

SynthesisofRuCy(Tz-4-P)library.[Ru(p-cymene)Cl2]2(RuCy,21.4mg,0.035mmol)wasdissolvedinDMSO(3.5mL,10mM).Ligandstocksolution(30µL,20mM)wasdispensedintoapolystyrene96wellplate,with30µLofRuCystocksolutionaddedtoeachwell,followedby60µLwater(giving120µLof5mMcomplexes).Theplatewasleftstationaryfor18hatr.t.,beforeanalysisbyLCMS.

SynthesisofRuCy(Tz-1-MP)library.[Ru(p-cymene)Cl2]2(RuCy,38.3mg,0.0625mmol)wasdissolvedinEtOH(25mL,2.5mM).Ligandstocksolution(60µL,20mM)wasdispensedinto700µLglassvials,with240µLofRuCystocksolutionaddedtoeachwell.Theplatewassealedandshakenatr.t.(600rpm)for48h,afterwhichtheEtOHwasevaporatedunderaflowofnitrogen,andthecompoundsdilutedwithDMSO(120µLof10mMcomplexes),beforeanalysisbyLCMS.

Generalprocedureforsynthesisoftargetligands.FSO2N3wassynthesizedasbeforeanddilutedwithDMFtoapprox.200mMin1:1MTBE:DMF(byvolume).Thedesiredamine(0.56mmol,1.0equiv.)waschargedintoa50mLpolypropyleneFalcontube,followedbyDMF(7mL).Sat.KHCO3solution(0.8mL,approx.2.4mmol,4equiv.inwater)wasadded,followedbyFSO2N3solution(1.05equiv.).Theresultingmixturewasstirredatr.t.for18h.Sodiumascorbatesolution(5.6mL,50mM,0.5equiv.inwater)wasadded,andthemixturewasstirredforafurther10min.7mLofDMFwasadded,followedbyalkyne(0.56mmol,1.0equiv.),then(BimH)3solution(0.7mL,20mM,2.5mol%inDMF)andCuSO4solution(0.7mL,20mM,2.5mol%inwater).Theresultingmixturewasstirredforafurther18hatr.t.ThecrudereactionmixturewasanalyzedbyLCMS,then100mLofdeionizedwaterwasadded.Themixturewasextractedwith3:1CHCl3:isopropylalcohol(3×30mL),followedbyEtOAc(2×30mL).Thecombinedorganiclayerswerewashedwith5wt%LiClsolution(5×25mL),dried(MgSO4),filtered,andthesolventevaporated.TheresiduewasredissolvedinEtOAc(40mL)andwashedwithmoreLiClsolution(3×20mL).Theorganiclayersweredried(MgSO4),filtered,andthesolventevaporatedtogivethecrudeproduct.Purificationwasachievedbyflashcolumnchromatography.

GeneralprocedureforsynthesisoftargetRecomplexes.Re(CO)5Cl(50mg,0.138mmol,1equiv.)andAgOTf(35.5mg,0.138mmol,1equiv.,Note:hydroscopic,light-sensitive)werechargedtoa5mLmicrowavevial,followedbyMeOH(2mL).Thevialwassealedandheatedinamicrowavereactorat120°Cfor30min.Thecolorlesssolutionwasfilteredthroughcelite(withanadditional1mLofMeOH)intoasecond5mLmicrowavevialcontainingthetargetligand(0.152mmol,1.1equiv.)and1-methylbenzimidazole(BeMeIm,20mg,0.152mmol,1.1equiv.).Thevialwassealedandheatedinamicrowavereactorat120°Cfor1h.Thesolventwasremovedtogivethecrudecompoundasapaleyellowsolid,whichwaspurifiedbycrystallization(approx.2mLDCM,layeredwithapprox.15mLn-hexane,andstoredat5°Covernight)orflashcolumnchromatography.

GeneralprocedureforsynthesisoftargetMncomplexes.Allreactionvesselsandcontainerswerecoveredinaluminiumfoilandhandledinthedarktopreventdegradationbylight.Mn(CO)5Br(91mg,0.33mmol,1equiv.)andAgOTf(85mg,0.33mmol,1equiv.,Note:hydroscopic)werechargedtoa10mLmicrowavevial,whichwasevacuatedandbackfilledwithN2.DryTHF(4.5mL)wasadded,andthevialwassealedunderN2andheatedat75°Cfor45min,afterwhichitwasallowedtocool.ThepressurewasreleasedbypiercingthesealwithaN2balloonwhencool,and2mLoftheyellowsolution(0.15mmolMncomplex)wasdecantedintoasecond10mLmicrowavevialcontainingtargetligand(0.155mmol,1.05equiv.)and1-methylbenzimidazole(BeMeIm,21mg,0.155mmol,1.05equiv.)underN2.Thevialwassealedandheatedat75°Cfor2h.Thesolventwasremovedinvacuotogivethecrudecompoundasayellowsolid,whichwaspurifiedbyflashcolumnchromatography.

GeneralprocedureforsynthesisofTargetIrCNcomplexes.Toa100mLroundbottomflaskfittedwithanaircondenser,IrCNdimer(40mg,0.037mmol,1equiv.)andtargetligand(0.074mmol,2equiv.)wereadded.CHCl3(30mL)andMeOH(10mL)wereadded,andtheresultingsolutionwasstirredrapidlyat40°Cfor18h.Thesolventwasremovedinvacuo,andtheresiduewaspurifiedbyflashcolumnchromatography.

GeneralprocedureforsynthesisoftargetIrCp*complexes.Toa7mLglassvial,di-μ-chloro-bis[chloro(pentamethylcyclopentadienyl)iridium(III)](40mg,0.05mmol,1equiv.)andtargetligand(0.10mmol,2equiv.)wereadded.DCM(2mL)andMeOH(2mLwereadded,andthereactionmixturestirredovernightatroomtemperature.Themixturewastransferredtoa20mLvialandthesolventwasevaporatedinvacuo.TheresiduewasdissolvedinDCM(2mL),towhichEt2O(10mL)wasslowlyaddedtoprecipitatethecomplex.ThefineprecipitatewasisolatedonaglasssinterandwashedwithEt2O(3×20mL).TheprecipitatewasdissolvedinDCM,collectedandthesolventremovedinvacuo.Et2O(2mL)wasaddedtotheresidueanddriedinvacuo,yieldingthetargetcomplexasayellowsolid.

Catalyticassays
GeneralprocedureforCoumarin-N3catalysisassay.Coumarin-N3(10mg,0.05mmol)andsodiumformate(34mg,0.5mmol)weredissolvedinamixtureof1:1tBuOH/1XPBSsolution(25mL)togiveasolutionof2mMcoumarin-N3.Inapolystyrene96wellplate,165μLof1:1tBuOH/1XPBSsolutionwasadded,followedbycoumarin-N3/sodiumformatemix(25μL).10μLofRuCycatalyststocksolutionat5mMwasaddedtoinitiatethereaction.Thereactionwasmonitoredbyfluorescence(generationofCoumarin-NH2)usingaplatereader(λex=350nm,λem=445nm)over21hat25°C,with2minofshaking(400rpm)and13mindelaybetweendatapoints.

Generalprocedureforharmalinecatalysisassay.IrCp*catalystsamplesweredilutedtoastocksolutionof750µM(50:50DMSO/H2O).Separately,stocksolutionsofharmaline(4.3mg,0.02mmol,2mM)andsodiumformate(136mg,2mmol,200mM)weredissolvedinmixturesof1:1tBuOH/1XPBSsolution(10mL).Inapolystyrene96wellplate,135uLof1:1tBuOH/1XPBSsolutionwasadded,followedbytheharmalinestocksolution(20µL,1equiv.),thensodiumformatestock(25µL,125equiv.),andfinally20uLofIrCp*catalyststocksolutionat750µMwasaddedtoinitiatethereaction.ThereactionwasmonitoredbyUV-Visabsorbance(consumptionofharmaline)usingaplatereader(λ=374nm)over48hat25°C,with2minofshaking(400rpm)and3mindelaybetweendatapoints.

Photophysicalmeasurements.5µLofeachcompoundintheIrCNlibrary(5mM)wasdilutedwith200µLDMSO.Ofthis,50µLwastakenanddilutedwithafurther150µLDMSOinapolystyrene96wellplate(31.3µM).TheUV-Visabsorbance(270–600nm,25flashes)andfluorescence(λex=406nm,λem=450–700nm,andλex=380nm,λem=450–700nm,25flashes)ofthesecompoundswerethenmeasuredusingaplatereader.

Biologicaltesting.Thefollowingbacterialstrainswereusedfortesting:EnterococcusfaecalisCCUG19916T(Grampositive),EnterococcusfaeciumCUG19434(Grampositive),StaphylococcusaureusCCUG19434(Grampositive),EscherichiacoliNCTC13476(Gramnegative).Bacterialstocksolutionswerepreparedasfollows.SinglebacterialcoloniesweregrownonTrypticSoyBroth(TSB,Grampositive)orMiller’sLBBroth(LB,E.coli)agar.Asinglecolonywasremovedandplacedintherelevantgrowthmedium(5mL,TSBorLB)andincubatedovernightat37°C(200rpm).100µLofthisovernightculturewasaddedto5mLofthesamegrowthmedium,andthedayculturewasincubatedat37°C(200rpm)untiltheOD600was0.4–0.9(~4h).AsampleofthisculturewasdilutedwithMuellerHinton(MH)brothtoOD600=0.022,atwhichpointthebacterialcultureswereaddedtoplatesfortesting.Foralltesting,sampleswereincubatedwithbacteriafor16–20hat37°C,afterwhichtheabsorbanceat600nmwasmeasuredusingaplatereader.AntibioticcontrolswereVancomycin(stocksolutionat1mg/mLinwater,MICbeginningat20µg/mL)forGrampositivebacteria,andPolymyxinB(stocksolutionat1mg/mLinwater,MICbeginningat20µg/mL)forE.coli.Unlessstatedotherwise,2biologicalrepeatspersamplewererecorded,witheachhaving2technicalrepeats.Polystyrene96wellplatesfromTPPandCytoOnewereusedinallcases(sterile,DNA,DNase,RNase,pyrogenfree).DMSOandothersolventswerekeptbelow5%volumeinthefinalsolutionsforbiologicaltesting.

Bacterialsingledoseresponseassay.Samplesweredilutedto750µMusingDMSO/H2O(togivea50:50stocksolution).Ofthesestocksolutions,20µL(for50µM)or5µL(for12.5µM)persamplewasaddedto96wellplates.MHgrowthmediawasaddedtoeachwell(makingupthevolumeto290µL),followedbytherelevantbacteriainMHgrowthmedia(10µL).Theplateswereincubatedfor16–20hat37°C,afterwhichtheabsorbanceat600nmwasmeasuredusingaplatereader.

Bacterialminimuminhibitoryconcentrationassay.StocksolutionsofsamplesweredilutedwithDMSO/H2O(togivea50:50stocksolution)sothat6uLofstocksolutioncanbeaddedto300µLofMHgrowthmediatogiveaninitialconcentrationofeither50µM(crudecompounds)or100µM(purecompounds).Thesesolutionswereseriallydiluted(150µLinto150µL)7times(crudecompounds)or11times(purecompounds)togivesolutionsof150µL.Tothese,5µLoftherelevantbacterialstrainwasaddedinMHgrowthmedia.Theplateswereincubatedfor16–20hat37°C,afterwhichtheabsorbanceat600nmwasmeasuredusingaplatereader.Purecompoundsweretestedwith2biologicalrepeatspersample,witheachhaving3technicalrepeats.FortestingwithPolymyxinBnonapeptide,asolutionof300µg/mLwasprepared,and5µLofthiswasaddedtoeachwellimmediatelybeforetheadditionofE.coli.

Toxicitytesting
Cellculture.Thehumanembryonickidney(HEK)293TcelllinewasculturedinDulbecco’sModifiedEagleMediumhighglucose(DMEM)(SigmaAldrich,USA)supplementedwith10%heat-inactivatedfoetalbovineserum(FBS),100μg/mLstreptomycinand100Units/mLpenicillin,and2mML-glutamine.Thecellsweremaintainedina5%CO2environment,at37°Candtheculturemediawerereplacedwithfreshmediaevery72hours.

Cytotoxicitystudiesoncrudelibraries.HEK-293Tcellswereseededat10000cellsperwellandincubatedovernighttoenableadhesion.Thereafter,thecellsweretreatedwitheitherthenegativecontrol(0.1%-0.28%DMSOdependingontheconcentrationofthecrudecomplexes)orthecrudecomplexesat50µMfor24h.Toquantifycellviability,thecellsweretreatedwiththe3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide(MTT)salt(ApolloScientific,UK)accordingtotheproceduredescribedbyMosmann71.Theabsorbanceat590nmwasmeasuredusingaTECANwellplatereaderinstrumentM1000.Twobiologicalrepeatsinduplicatewereperformedforthisstudy,andtherecordedabsorbanceswereadjustedfortherespectivecrudesandgrowthmedia.

Determinationofthecytotoxicconcentration50%(CC50).HEK-293Tcellswereseededat10000cellsperwellandincubatedovernighttoallowadhesion.5mMstocksolutionsofpurecompoundswerepreparedin1:1DMSO:PBS1X.Ina1.5mL96welldilutionplate,startingconcentrationsof150µMand200µMofeachcompoundweremadeingrowthmedia(1mL).500uLofthesewereserialdilutedinto500uLofmedia11times.500uLofmediawasthenaddedtoeachwelladdedtomake1mLofeachconcentration(startingat75µMand100µM).100uLofthesesolutionswereaddedintowellscontainingthepre-countedcellsandtreatedfor24h.Thereafter,theMTTassaywasusedtoquantifyviablecellsandatleastthreebiologicalrepeatsintriplicatewereconductedfromwhichtheCC50wasdeterminedusingGraphPadPrismv8.0.2(GraphPadSoftware,California,USA).Theabsorbanceat590nmwasmeasuredusingaTECANwellplatereaderinstrumentM1000.4biologicalrepeatsintriplicateweredoneforthisstudy(12datapoints).FortheMncomplexes,2biologicalrepeatsintriplicate(6datapoints)wereused.

Haemolysis.BloodsampleswereobtainedfromhealthyvolunteersthroughHullYorkMedicalSchool.Thehaemolysisassaywasadaptedfromapreviouslyreportedprocedure72.Wholeblood(5mL)wascentrifuged(ThermoScientificSL8R)at4500rpmfor1hat4°C,andtheplasmawasdiscarded.Theremaininghumanredbloodcell(hRBC)pelletwaswashedthreetimeswithPBS1X(pH7.4):foreachwash,PBSwasaddedupto15mL,followedbycentrifugationat4500rpmfor1hat4°C.Afterthefinalwash,thecellswereresuspendedinPBStoafinalvolumeof35mL.Thestocksolutionsforthesamplestobetestedwere2.5mMinPBS:DMSO(60:40).ForthedeterminationofHC₅₀andHC₁₀values,testcompoundsweredilutedinPBStogiveaconcentrationrangefrom200µMdownto5µM,givingatotalvolumeof125µL.Dilutionswerepreparedin20µMdecrementswiththefinalpointsat10and5µM.Eachplateincludedablankmediumcontrol(PBS)andahaemolyticactivitycontrol(1%SDSinPBS).ThehRBCsuspension(125µL)wasincubatedwiththesamplesinPBSinaV-shaped96-wellplatefor4hat25°C.Afterincubation,60μLofthesupernatantwascarefullypipettedtoaflatbottom,clear96-wellplate.Haemolysiswasmeasuredbyabsorbanceat540nmusingaplatereader(TECANM1000).ThepercentageofhaemolysisateachconcentrationwasdeterminedandtheHC10andHC50werecalculatedbyinhibitorvs.normalizedresponsefit(Prism).Theminimumhaemolyticconcentration(MHC)wasdeterminedbyvisualassessmentofthewellsafterincubation.TheMHCwasdefinedasthelowestcompoundconcentrationatwhichvisiblehaemolysis(redcolorationofthesupernatantand/orlossofintactredpellet)couldbeobservedcomparedtothePBScontrol.Eachexperimentwasrepeatedintriplicate(3biologicaland3statisticalreplicates).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript under review aims to develop a high-throughput combinatorial click chemistry platform for the synthesis of triazole-based metal complexes, followed by their biological evaluation as antimicrobial agents. The general idea of merging modular synthetic chemistry with biological screening remains an exciting and productive avenue in drug discovery. However, the present study suffers from fundamental scientific and conceptual weaknesses that undermine its reliability, novelty, and overall significance. 1) Compound Purity and Analytical Standards: Perhaps the most critical and immediately disqualifying issue in the manuscript is the lack of purity of the compounds under investigation. The authors proceed with photophysical measurements and biological activity screening despite demonstrating that the tested compounds are of sufficient chemical purity. This is not a minor technical oversight; it goes to the heart of the study’s scientific validity. As established across the scientific community and explicitly stated in the author guidelines of major publishing houses (e.g., Springer, Wiley, ACS, RSC), a minimum compound purity of 95% is required for any molecule subjected to biological assays. This requirement exists for good reason: numerous research studies have shown that even small levels of impurities can generate significant biological effects, thereby misleading the interpretation of structure–activity relationships and potentially invalidating entire studies. It is not enough to simply state that a compound was synthesized and used. Proof of purity (e.g., HPLC, elemental analysis, clean NMR spectra, and LC-MS) is absolutely essential! In this manuscript, the authors present no evidence that any of the tested compounds reach the 95% purity threshold, and in fact, the data they do present indicate that most compounds fall significantly short of this mark. As a result, the biological and photophysical data must be viewed as unreliable and cannot be used to support any meaningful scientific conclusion. Before submission to any journal, the authors must purify all compounds to the appropriate standard, provide full characterization data, and then repeat all experimental evaluations. Failure to do so will continue to render this study unsuitable for any reputable publication. 2) Synthetic Methodology and Combinatorial Strategy: The manuscript claims to offer a high-throughput, click-chemistry-based strategy for the rapid generation of a compound library. However, a closer examination of the synthetic procedures reveals a complex, multistep process that includes repeated reagent additions and sequential manipulations. This raises serious doubts about the claim that the method is combinatorial or high-throughput in practice. True combinatorial chemistry, particularly when described as “high-throughput”, implies simple, robust, one-pot procedures that minimize the number of reaction steps, maximize yield and reproducibility, and require minimal purification. The methodology described in the current manuscript does not meet these criteria. Furthermore, the click chemistry component is not exploited to its full synthetic potential. Although the CuAAC reaction is well-known for its efficiency and modularity, its use here appears limited to decorating pre-existing metal scaffolds rather than generating fundamentally new or diverse structures. This further contributes to the lack of novelty and weakens the overall impact of the synthetic approach. 3) Chemical Diversity and Novelty: A major weakness of the manuscript is the poor chemical diversity within the reported library. Nearly all of the compounds are built upon well-known and widely studied metal scaffolds, including: Rhenium tricarbonyl complexes (i.e., Inorg. Chem. 2019, 58, 3895−3909), Manganese tricarbonyl derivatives (i.e., Chem. Sci. 2024, 15, 3907–3919), Cyclometalated iridium complexes (i.e., Angew. Chem. Int. Ed. 2024, 63, e202401808), Ruthenium arene compounds (i.e., J. Med. Chem. 2014, 57, 6043−6059). Only a fraction of prior works are referenced in the current manuscript, which raises concerns about a lack of awareness of the state-of-the-art. Omitting key citations not only diminishes the scholarly quality of the manuscript, but also misleadingly implies novelty where there is little to none. Moreover, even within these established classes, the structural variations introduced are minimal. This undermines the central premise of combinatorial exploration, namely, that the library should explore broad chemical space to uncover new structure–activity relationships. Instead, the authors present a narrow, homogeneous collection of analogs whose differences are unlikely to yield substantially different biological outcomes. Without significant scaffold innovation, ligand diversification, or new coordination geometries, the manuscript offers little that is new or original, and therefore does not meet the standards expected of high-impact publications such as Nature Communications. 4) Biological Evaluation: Limited Depth and Mechanistic Insight: While the study does report antimicrobial activity for the tested compounds, the biological data are limited to simple inhibition assays. There is no mechanistic insight, no target identification and no attempt to understand the mode of action of the compounds. This is a missed opportunity and severely limits the biological relevance and translational value of the findings. High-impact journals require more than just activity data; they demand a deeper understanding of how compounds function at the cellular and molecular level. Without such mechanistic work, even promising activity is insufficient to justify publication in a top-tier venue. Additionally, the lack of proper controls, such as comparisons with known active metal complexes or the absence of structure–activity relationship analysis, further weakens the biological conclusions. Again, the low purity of the compounds severely undermines any interpretations of the data, raising the real possibility that observed activity may be due to unknown impurities. This study raises several interesting ideas, but unfortunately fails in execution on nearly every critical metric: Lack of compound purity renders all biological and photophysical data unreliable, Synthetic methodology is not truly combinatorial or high-throughput., Minimal novelty or diversity in chemical structures with known scaffolds, and superficial biological evaluation with no mechanistic insight. In its current form, the manuscript does not meet the standards required for publication in Nature Communications or any peer-reviewed chemistry or medicinal chemistry journal. I strongly recommend that the authors revisit the core elements of their study, perform a complete overhaul of the synthesis and characterization workflow, and critically engage with the existing literature. Only then might the work have the potential to make a meaningful contribution to the field.", "reviewer_2": "This manuscript by Frei and coworkers provides the access to a high number of novel metal complexes, using traditional click chemistry and SuFEx chemistry. The innovation in terms of chemistry is quite limited, however, the substantial number of complexes is impressive. Although, only few selected ones have been re-synthesized. From the point of view of this reviewer, it is a shame that more emphasis was not dedicated to catalytic properties, rather than antibacterial properties, where many challenges need to be overcome if these were to be used clinically. This includes the cost of at least some of these metals, which appear to be a limiting factor, that should be discussed. Further, a realistic discussion of the potential of such complexes as antimicrobials should be added, including stability in vivo, type of infection (e.g., systemic vs. topical), delivery (e.g., injectable, oral, creme). Finally, both the click chemistry and the SuFEx chemistry is not discussed in sufficient detail and seminal citations are missing.", "reviewer_3": "The manuscript presents a combinatorial methodology for synthesizing extensive libraries of pyridyltriazole bidentate ligands via CuAAC click chemistry. These ligands were coordinated with five different metal scaffolds under mild conditions, resulting in 672 novel metal complexes. The authors have assessed the photocatalytic potential of selected libraries. Notably, the entire set of metal complexes was screened for antibacterial activity and cytotoxicity using high-throughput methods, leading to the identification of six promising compounds. These hits were resynthesized and demonstrated nanomolar-range antibacterial activity against Gram-positive bacteria. Overall, the results are compelling, and the study is well-executed and clearly presented. I support the publication of this work in Nature Communications. However, there are a few issues that need to be addressed by the authors before acceptance. 1. The manuscript employs a combinatorial approach using metals like Ir, Ru, Re, and Mn. While the rationale for most metal choices is understandable, the specific reasoning behind selecting manganese (Mn) for this study is unclear. Could the authors provide more justification for its inclusion? 2. The purity and identity of the compounds were primarily assessed by LC-MS. However, in several HPLC chromatograms, the product peak appears alongside additional peaks that may represent impurities. These impurities could potentially influence biological or catalytic activity (e.g., antibacterial effects or photocatalysis). Have the authors considered this possibility in their analysis while screening of the compounds? 3. The manuscript states that “The lowest MIC measured was 0.39 µM for 2 compounds...” in the IrCN libraries. However, according to Figure 7C (MIC distribution), four compounds appear to exhibit this MIC value. The authors should clarify this inconsistency. 4. In the Mn(CO)₃ library, compounds M19Y1 and M22Y1 are described as the purest based on LC-MS data. Could the authors elaborate on how these were deemed the purest? 5. While cytotoxicity was assessed using HEK293T cells, no data on hemolytic activity of the hit compounds is presented. To better evaluate bacterial selectivity and potential safety, hemolytic activity should also be investigated. 6. Given the known luminescent properties of IrCN complexes, the authors could explore their application in bacterial imaging. This would help determine whether these compounds are internalized by bacterial cells. 7. The manuscript states that “Even after 18 h of irradiation, no conversion >2% was achieved” under “Catalysis.” However, Figure S4 shows a maximum conversion of only 0.54%. This statement should be corrected to reflect the actual data. 8. The catalytic study using Coumarin-N₃, while interesting, appears disconnected from the antibacterial activity. No prodrug activation has been demonstrated in bacterial cells or extracellular media. This section could be omitted or moved to the Supporting Information to maintain focus and clarity. 9. The authors should provide possible mechanistic or structural explanations as to why M11Y2, M13Y2, and M24Y2 exhibited superior catalytic activity compared to their Ru-based analogues. 10. In many parts of the text, only the figure number is mentioned without specifying the relevant panel (e.g., A, B, or C). This is especially problematic in figures that present multiple experiments, such as Figure 6. For clarity, the authors should refer to specific panels—for example, “Figure 6(B–D)” for the transfer hydrogenation (TH) catalysis by RuCy libraries. 11. What is the rationale behind reporting absorbance values in various solvents in Table S2? While it is expected that absorbance values will vary depending on the solvent used, why are negative absorbance readings observed for M20Y3 in DMSO, M12Y1 in DMSO/H₂O (1:1) and PBS 1X, as well as for M8Y4 and M22Y1 in certain solvents? Do these negative values indicate that the complexes are unstable or degrading in these solvent systems?"}, "author_response": "1) Compound Purity and Analytical Standards: The reviewer states that ‘a minimum compound purity of 95% is required for any molecule subjected to biological assays’ and that ‘even small levels of impurities can generate significant biological effects’. We agree that for final compounds such a degree of purity is certainly warranted and indeed we have provided 1H-13C NMR, HR-MS and LC-MS data to prove the purity for all resynthesized hit-compounds in our manuscript and SI. However, to provide this for all compounds reported in this study is impractical and would defeat the purpose of our approach. The goal of the study was to access a large number of compounds quickly and evaluate them efficiently for a desired property. If we had set out to purify and fully characterize all compounds this would take an unfeasible amount of time. Additionally, most compounds will not have ideal properties so most of that time would be wasted. The approach we are following is not novel and has been widely applied in both academia and industry for decades, with it recently having been referred to as “Direct-to-Biology” (D2B). Indeed, there are dozens of high-impact papers published in established journals. We, and others across both academia and industry, have shown in numerous studies that crude compounds can indeed serve as proxies to identify compounds with promising properties and that the readouts in the assays of our work for purified compounds align very well with the crude mixtures. We have collated a list of high-impact journal publications in which the same or a similar approach was taken, some of which can be found in prestigious Nature journals (a seletion of these have been added to the revised manuscript): [List of 14 citations omitted for brevity]. In summary, this approach (Direct-to-Biology) is a valid, known and evolving methodology that is amenable to high-throughput screening. As such, the requirement by the reviewer to ‘purify all compounds to the appropriate standard, provide full characterization data, and then repeat all experimental evaluations’ is an impractical suggestion. The core hypothesis of our work is that crude reaction mixtures of carefully designed assembly reactions can be used to quickly evaluate large libraries of compounds and identify promising hits quickly. There is of course a chance for false positives, but we address this by testing all building blocks which generally are not biologically active nor possess catalytic activity as well as resynthesizing, purifying, characterising and re-testing selected hit compounds. Indeed, in some cases the combinatorial reactions are not fully clean. However, in the majority of cases, the target compound has formed with at least 50% conversion. In the biological testing there are four possible scenarios: Target Compound and Impurities are active: In this case the compound will be registered as active and if the activity level is high, it will be resynthesized and purified, in which case it will become clear if activity is from the putative compound. Given that most organic and inorganic compounds are not active against bacteria, we expect this scenario to only occur in a minority of all cases (Frei et al. Chem. Sci. 2020 DOI: https://doi.org/10.1039/C9SC06460E). Target Compound is active, impurities are inactive: Given that in most cases 50% or more of the crude mixtures is the target compound, the antibacterial assays will still pick up the activity of the target compound and any activity observed will be due to the latter. If the activity level is high, resynthesis and purification will verify this. Target Compound is inactive, impurity is active: This would constitute a real false positive. If the impurity has strong activity this could lead to the resynthesis of a compound that then shows no activity. This has not occurred in our work so far (Scaccaglia et al., Chem. Sci., 2024,15, 3907-3919;d3sc05326a; Orsi et al., Angew. Chem. Int. Ed. 2024, 63, e202317901;https://doi.org/10.1002/anie.202317901) and as such we deem it unlikely. Additionally, if a singular impurity showed very high activity that could be an interesting compound to further investigate as well. Target Compound and impurities are inactive: Based on historical data (most molecules are inactive), this is the most common scenario, and in this case the compound is not of interest, so we do not miss any of these compounds. To better clarify our approach, we have added a paragraph supporting the “Direct-to-biology” into the manuscript. The comments about using crudes for photophysical effects can be addressed similarly. We tested the two resynthesized (and pure) IrCN complexes for fluorescence and absorbance, and the emission spectra match the crude samples. This allows us to assume with confidence that the fluorescent species are indeed the target complexes. We do acknowledge some of the issues with the approach in the revised manuscript but carefully explain the mitigations that can and were undertaken in our study. Correction: To contextualize the approach that we have taken, we have included a paragraph in the manuscript introduction that explains “Direct-to-Biology”, using libraries of not purified compounds as a screening technique. This should leave no doubt as to the validity of our study and makes clear the approach we have taken: [Paragraph text included in manuscript]. 2) Synthetic Methodology and Combinatorial Strategy: We disagree with the reviewer’s assessment that our approach does not represent a high-throughput combinatorial approach. Our synthetic platform, as described, could readily be expanded to prepare thousands of novel complexes with minimal effort. Instead of scale, we chose to focus on breadth and prepared novel metal complexes across two ligand and five metal scaffolds. While our reactions have three distinct steps, these can all be done in one pot and require a minimal number of liquid transfers, which we demonstrate can be handled by an accessible automated liquid handling robot (an OT2 as used in this work can be bought for less than 20,000 USD). We are unsure what the reviewer is refereeing to in saying ‘the click chemistry component is not exploited to its full synthetic potential’. We have shown that we can prepare two distinct types of triazole-base ligands from chemically diverse starting materials (including complex organic scaffolds such as ampicillin) in a 96 well plate array with good conversions and coordinate these to five distinct metal scaffolds. By nature of design, we chose metal scaffolds where we expected the coordination chemistry to work reliably as there has not been any literature precedent for the high-throughput preparation of the reported compounds. It is of course our hope that other researchers will adopt this methodology to expand the scope of the work even further. We would like to point out that our work goes significantly beyond what has been done before. Previous work focused on individual metals and generally either applied Schiff-base type ligands or commercially available polypyridyl scaffolds. Our work both introduces two entirely new scaffolds to the combinatorial chemistry approach and applies it to five different metal scaffolds. Again, we then evaluate a series of properties of these compounds again going well beyond what has generally been done in previous work. 3) Chemical Diversity and Novelty: We have certainly noted other combinatorial metal complex approaches, indeed we have published a review article on ‘High-Throughput Combinatorial Metal Complex Synthesis’. (10.1002/anie.202420204) However, none of these previous studies on combinatorial synthesis utilize the triazole-type of ligand reported in our work. As mentioned before, the choice of well-behaved metal scaffolds was by design because a) we wanted to ensure that ligand coordination would work efficiently across many different ligands under 96 well plate conditions and b) there was previous data to indicate that these compound classes had promising properties as catalysts, luminophore and antimicrobial compounds. We strongly disagree with the notion that the modifications introduced into our ligands ‘are unlikely to yield substantially different biological outcomes’. Indeed, this is directly contradicted by the data reported in the manuscript and in any medicinal chemistry paper. We clearly show that small changes within the ligands can result in large differences in properties and our approach enables the preparation of larger libraries which in turn make it possible to chart these properties to structural modifications. To highlight the insights that can be gained we have expanded the Structure-Activity Analysis (SAR) in our manuscript. This involves calculating and observing trends between observed physical and biological properties with calculated parameters such as cLogP which are commonly used in analysis of drug molecules. More detail and graphs have been added to the SI to support this. To further validate that SAR insights can be gained from this approach we have trained a Support Vector Machine ML Model on the antibacterial activity of the rhenium and iridium complexes. The model results in a remarkably good fit and we were able to extract relevant substructure features that correlate with active compounds. The extended SAR and ML discussion has been added to the manuscript and the Supporting Information: [Detailed SAR/ML discussion included]. 4) Biological Evaluation: While we thank the Reviewer for their comments, we strongly disagree with them. Firstly, we re-iterate that the focus of this work is the novel methodology for accessing large modular libraries of transition metal complexes across five different metal scaffolds. We then show that these libraries can be evaluated for their properties in high-throughput “Direct-to-Biology” assays, enabling the rapid identification of promising compounds amongst libraries of hundreds (and possibly thousands). We then further confirm these assays by completing resynthesis, purification and full characterization of the hit compounds. Identifying the biological mechanism of action of a single compound represents a multi-year project and successful projects on individual molecules in this area regularly result in stand-alone articles in the highest tiers of journals. Secondly, we note that understanding the mechanism of action is not an FDA requirement for drug approval. What is required is for a compound to be shown to be efficacious and safe. Indeed, our best compound has a highly promising therapeutic index and is currently undergoing further studies. While it is a core interest of our group to investigate the mode of action of novel metalloantibiotics (Özsan et al. bioRxiv 2025 DOI:10.1101/2025.05.30.657029v1) and we will look to develop our lead compounds further, this clearly goes beyond the scope of this work. [Responses to Reviewer 2 and 3 included in the full text]."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The submitted manuscript represents a revised version of a previously evaluated study. Although the authors have provided additional details and have made certain clarifications in response to earlier reviewer comments, the major weaknesses identified in the initial submission remain unresolved. These deficiencies are not minor technical issues but rather fundamental scientific flaws that directly compromise the validity and credibility of the entire work. The most serious and immediately disqualifying issue concerns the purity of the compounds under investigation. The authors have carried out photophysical measurements and biological activity assays using samples that are demonstrably impure. In fact, based on the analytical information provided, it appears that several of the tested compounds contain impurity levels exceeding 50%. Proceeding with experimental work on such inadequately characterized materials is scientifically indefensible. Any data derived from these samples—whether photophysical, chemical, or biological—cannot be considered reliable or interpretable in a meaningful way. This issue strikes at the very core of scientific rigor and reproducibility. Across the chemical and biological sciences, it is an established and non-negotiable standard that any compound subjected to biological evaluation must possess a minimum chemical purity of 95%. This requirement is explicitly stated in the author guidelines of all major publishing houses, including Springer, Wiley, ACS, and RSC. The rationale behind this standard is clear and well-documented: even trace impurities can significantly alter observed biological effects, confounding the interpretation of structure–activity relationships and leading to erroneous conclusions. To disregard this principle is to undermine the integrity of the scientific process itself. In the present manuscript, the authors fail to provide any convincing evidence that the compounds used meet the accepted purity threshold. Essential analytical data—such as clean NMR spectra, LC-MS chromatograms, HPLC purity profiles, and elemental analysis—are either missing, incomplete, or clearly indicate that the samples are mixtures rather than pure compounds. Without proper characterization, it is impossible to assign the observed biological or photophysical properties to the intended molecules. The reported results must therefore be regarded as unreliable, non-reproducible, and scientifically meaningless. Furthermore, the authors appear to have proceeded with biological screening and photophysical measurements despite being aware of the inadequate purity of their materials. This represents a serious lapse in scientific judgment. It is the responsibility of every researcher to ensure that all data presented in a manuscript are based on rigorously characterized and validated materials. Failure to do so not only invalidates the specific findings of this work but also risks misleading future researchers and wasting scientific resources. Before this study could be reconsidered for publication, the authors would need to undertake a complete re-evaluation of their experimental approach. Specifically, they must: 1) Purify all compounds to at least 95% chemical purity, verified by appropriate analytical methods. 2) Provide full characterization data, including high-quality NMR spectra, LC-MS and HPLC purity analyses, and elemental analysis where applicable. 3) Repeat all photophysical and biological experiments using the verified pure compounds. Only after these steps are completed would the results be scientifically interpretable and potentially suitable for publication. In its current form, the manuscript fails to meet even the minimum standards of scientific reliability and integrity expected in the field. The core experimental data are invalidated by the use of impure materials, and the conclusions drawn from these data are therefore unsound. For these reasons, I find the manuscript wholly unsuitable for publication, particularly in a high-impact journal such as Nature Communications. In conclusion, while the authors have made certain revisions, the fundamental flaws remain unaddressed and continue to undermine the scientific foundation of the study. The lack of compound purity invalidates the reported findings and renders the entire work scientifically unreliable. I therefore strongly recommend that this manuscript be rejected.", "reviewer_2": "No further comments.", "reviewer_3": "The authors have addressed my concerns. I am satisfied with the revision work performed by the authors. The revision works have improved the quality of this manuscript. I recommend the publication as it is."}, "author_response": ""}]

Decision letter
[Note from the Editor: Reviewer #3 was also asked to assess the response to Reviewer #1. In comments to the editorial office, Reviewer #3 stated they were convinced by the response and that the manuscript is now suitable for publication.]