Interfacial Manganese‐Doping in CsPbBr3 Nanoplatelets by Employing a Molecular Shuttle

Abstract Mn‐doping in cesium lead halide perovskite nanoplatelets (NPls) is of particular importance where strong quantum confinement plays a significant role towards the exciton–dopant coupling. In this work, we report an immiscible bi‐phasic strategy for post‐synthetic Mn‐doping of CsPbX3 (X=Br, Cl) NPls. A systematic study shows that electron‐donating oleylamine acts as a shuttle ligand to transport MnX2 through the water–hexane interface and deliver it to the NPls. The halide anion also plays an essential role in maintaining an appropriate radius of Mn2+ and thus fulfilling the octahedral factor required for the formation of perovskite crystals. By varying the thickness of parent NPls, we can tune the dopant incorporation and, consequently, the exciton‐to‐dopant energy transfer process in doped NPls. Time‐resolved optical measurements offer a detailed insight into the exciton‐to‐dopant energy transfer process. This new approach for post‐synthetic cation doping paves a way towards exploring the cation exchange process in several other halide perovskites at the polar–nonpolar interface.

Preparation of Cs-Oleate (Cs-OA) solution.
To prepare Cs-OA solution, 0.16 g of Cs2CO3, 0.5 mL of dried Oleic acid (OA) and 8 mL of ODE were added into a 50 ml three-neck flask. The mixture was degassed under vacuum at 120 o C for 30 min, and then the temperature was increased to 150 o C under N2 atmosphere. After the solid phase was completely dissolved, the temperature was set back to 120 o C for further use.

Synthesis of CsPbBr3
NPls. The CsPbBr3 NPls were prepared following our previously reported literature. [1] In a typical synthesis of CsPbBr3 NPls, 0.069 mg (0.188 mmol) of PbBr2 was added into a 50 mL three-neck flask containing 0.5 mL of OA, 0.5 mL of OLA and 5 mL of ODE. The mixture was degassed under vacuum at 120 o C for 20 min and then changed to N2 atmosphere. When the PbBr2 was completely dissolved, the solution was cooled to room temperature. Subsequently, 0.4 mL of hot Cs-OA was injected into the solution. The mixture was heated up under an N2 atmosphere. Once the temperature reached 100 o C (150 o C or 180 o C), the reaction was quenched by an ice-water bath. The product was separated by centrifugation at the speed of 8000 rpm for 5 min. The precipitate was redispersed into 10 mL of hexane. To get the final product, the hexane solution was centrifuged at 3000 rpm for 5 min, and then the precipitate was discarded.

Mn doping process in One-step two-phase post-synthetic (OPS) method.
In a typical experiment, several concentrations of manganese salt in 50 L of water was mixed with 1 mL of as-prepared CsPbBr3 NPls and 1 mL of hexane. Subsequently, the two-phase mixed solution was subjected to stirring at the speed of 400 rpm for 1 h at ambient conditions. Then after waiting for a minute before there is a phase separation, the hexane layer was pipetted out from the upper phase. Generally, this step is not time-sensitive, however, prolonged mixing (over days) between the two phases can destroy the NPls to some extent (see Fig. S12). Furthermore, if the stirring is continued beyond 1 hr, it takes several days (~17 days) for complete degradation of the perovskite, with negligible PL emission. Therefore, it is better to separate the phases soon after the reaction. The hexane phase was then transferred to a centrifuge tube and treated by centrifugation at the speed of 9000 rpm for 5.
The precipitate was discarded, and the final product will be in hexane.

Mn doping process in two-step two-phase post-synthetic (TPS) method.
In this method, the Mn doping process needs two steps. Firstly, 50 L saturated aqueous solution of a manganese salt was added into 2 mL of hexane which contains 10 L of OLA. The mixed solution was subjected to oscillation by a vortex for 30 seconds at vigorous conditions. Then, the hexane solution on the upper phase was transferred to a centrifuge tube, similar to section 1.4, and treated by centrifugation at the speed of 9000 rpm for 5 min. The hexane solution with MnX2 on the upper phase was retained for further use, and the precipitate was discarded. Next, 1 mL of hexane with MnX2 prepared in the first step was mixed with 1 ml as-prepared CsPbBr3 NPls. The Mn-doped CsPbBr3 NPls will generate immediately. The mixture could be stirred for a certain time to make sure the reaction is completed.

Transmission electron microscopy (TEM).
Scanning transmission electron microscopy in high-angle annular dark-field mode was performed with a probe corrected FEI Titan Themis operating at 300 kV (Department of Chemistry, LMU Munich). TEM specimen was prepared on a carbon-coated Cu grid by dropping 10 L of dilute colloidal dispersions on each sample.

Electron Paramagnetic Resonance (EPR) spectroscopy.
The presence of Mn in the doped NPls is determined by an EPR spectroscopy from ADANI Spinscan X (2020 model). The conditions are used as following: 20mW, 9.4506 GHz. To prepare samples for the EPR measurement, first the prepared Mn-doped NPls (via OPS method) was precipitated with an anti-solvent (ethyl acetate) and collected after centrifugation, which would remove the undoped Mn ions in the solution. To get a sufficient amount of samples for measurements, 10 typical batches (NPls in 10 ml hexane) of the two doping concentrations were made and collected together. The solid sample was then filled into the quartz tube for EPR measurement. The EPR signals of the empty tube were first taken as the baseline.

Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES).
The amount of Mn-doping in the NPls is determined by ICP-OES measurement from Varian Vista RL simultaneous spectrometer with autosampler. To prepare samples for the ICP-OES measurement, first the prepared Mn-doped NPls (4M and 7M) (via OPS method) were precipitated with an anti-solvent ethyl acetate and collected after centrifugation, which would remove the undoped Mn ions in the solution. To get a sufficient amount of samples for measurements, 5 typical batches (NPls in 10 ml hexane) of the two doping concentrations were made and collected together. Then the solid doped NPls were dissolved in 400 μL aqua regia (freshly made and stirred for over 30 min) under strong sonication, which were further diluted to 20 ml aqueous solutions. The samples were filtrated before measured in the ICP-OES equipment.

UV-Vis absorption and Photoluminescence (PL) spectroscopy.
The absorption spectra were measured by a Cary 5000 UV-Vis-NIR spectrophotometer. A Horiba Jobin Yvon Fluorolog-3 FL3-22 spectrometer with a 450W Xe lamp was used to acquire the photoluminescence spectra.

Time-resolved fluorescence spectroscopy.
A homemade device with a white light laser (NKT SuperK Extreme EXR-20), extended with an EXTEND-UV box, was used to perform the time-resolved fluorescence measurements. The samples were excited with 350 nm laser. The signal was detected with a Princeton Instruments monochromator fiber connected to a low-noise Avalanche Photodiode from Excelitas.

Femtosecond transient absorption spectroscopy. A custom-built transient
absorption setup from Newport was used for the ultrafast transient absorption measurement, similar to our earlier work. [2] A femtosecond multi-pass Ti: Sapphire regenerative amplifier laser system (Libra HE+, Coherent Inc.) produces ~100 fs laser pulse having 1 kHz repetition rate at 800 nm. A fraction of this laser pulse is frequency doubled using a β-barium borate (BBO) crystal to produce the pump beam at 400 nm for the sample excitation. Another fraction of the laser pulse (~1 J) is used to generate the white-light probe pulse by focusing part of the initial 800-nm beam through a 2-mm-thick motorized CaF2 crystal. The pump and the probe pulses are spatially and temporally overlapped at the sample, with the pump pulse passing through at 0.5 kHz chopper to read out the changes in probe transmission for every pump-probe cycle. A motorized optical delay line is placed in the probe arm to offer a time delay between the two pulses. The NPls dispersed in hexane were kept in a 2-mm cuvette for the pump-probe experiment. The average number of excitons were kept low enough (<<1) to avoid the multi-exciton phenomenon.

Possible Mechanism of Mn-incorporation into the NPls as a function of thickness.
To understand the underlying mechanism of Mn incorporation into the NPls as a function of the thickness of parent NPls, we performed a detailed investigation of the stability of the undoped and doped NPls prepared at 100 and 180 o C, by probing the PL spectra. As shown in Fig. S10-12, the undoped NPls prepared at 100 o C is only stable for a few days (<10 days), while the one prepared at 180 o C is stable for more than 10 days, as the PL spectra remain unchanged on the 10 th day. The same phenomenon has also been observed for the corresponding Mn-doped NPls. Here the intensity of the Mn PL for the doped NPls obtained from the Un-100 NPls (named Mn-100) drops significantly at the 10 th day while that for the Mn-180 NPls remains quite similar to day 1, albeit a slight blue shift in the Mn PL is observed for both Mn-100 and Mn-180 NPls (see section 3.2). One possible reason for the better stability of the NPls prepared at a higher reaction temperature (180 o C) corresponds to thicker NPls (5 ML) due to better surface passivation. [3] This directs that most of the ligands (both OA and OLA) are engaged towards the surface passivation of the NPls prepared at a higher reaction temperature. As revealed in the main text, the post-synthetic method developed in this work is based on the shuttling effect of the OLA, which is liable for the MnX2-transport at the water-hexane interface. Due to better surface passivation, the number of available shuttling ligands is limited in the hexane solution, leading to low incorporation of Mnion into the 5 ML NPls prepared at 180 o C.
Conversely, the NPls prepared at 100 o C showed lower stability due to poorer surface passivation. This not only leads to additional availability of the shuttling ligands in the solution but also opens more room for Mn-ion incorporation through poorer surface passivation, eventually leads to more Mn-ion incorporation into the NPls (i.e. Mn-100). The additional available OLA ligand plays the shuttling role, which can be verified from the fact that if the amount of OLA is increased in the hexane solution (as described in Fig. 2a), the incorporation of Mn-ion into the NPls increases (see Fig. S6).
As a result, one can conclude that the amount of the Mn-ion incorporation into the NPls in the post-synthetic method described in this work depends on the number of available shuttling ligands (here OLA) in the nonpolar solvent. Moreover, as Mn-100 (7M) NPls are less passivated compared to Mn-180 (7M) NPls, this indicates the organic ligand may plays an essential role in the energy transfer process in the two system, as described later.
One should also note that, a small side band along with the main exciton PL peak is observed in some of the samples (Fig. S5, S8, S9). Most probably, these are due to formation of different size/thickness NPls, which forms possibly due to different solvent mixture used in the post-synthetic doping.

Mn PL tunability as a function of NPls thickness.
The atomic like Mn PL in the doped NCs or NPls is supposed to appear ~600 nm (2.1 eV), independent of the host NCs/NPls. However, it is also reported the Mn PL peak position tunability upto ~55 nm, starting from ~585 nm to ~640 nm, in perovskite nanocrystals [4] . The atomic like 4 T1-6 A1 Mn PL peak position tunability depends on a few factors [5] : 1) ligand field strength,

2) Mn -Mn interaction,
3) doping induced lattice contraction effect in the crystal field strength, etc.
In the present investigation, the Mn PL peak position appears ~618 nm for the Mn-180 (7M) NPls. On a closer inspection suggests the Mn PL peak position blue-shifted upon reduction of the parent NPls thickness; for example, the Mn PL appears at ~608 nm and ~605 nm for Mn-150 (7M) and Mn-100 (7M) NPls, respectively (see Fig. 3b). On the contrary, the Mn PL peak position is independent of Mn amount for a particular thickness, e.g. it always appears ~618 nm for all Mn-180 NPls (see Fig. 1g), indicating the Mn -Mn interaction and doping induced lattice contraction may not be the underlying reason for Mn PL position tunability in the present case. We believe the Mn PL peak position tunability in the present case may be related to the ligand field strength.
Here the Mn-dopant in the NPls with a lower thickness (e.g. Mn-100 NPls) faces a higher ligand field interaction from the surrounding shuttle ligands due to additional available shuttle ligands. This eventually leads to more splitting of the 4 T1-6 A1 energy, causing the blue shift from Mn-180 to Mn-100 NPls.
Another thing also needs to be considered: upon Mn-halide saturated aqueous solution addition, a certain amount of Mn-halide shuttled to hexane via OLA, all of which (i.e. Mn-OLA complex) indeed does not lead to doping in the NPls. As a result, the doping process still may continue after several days, even after separating the two phases. This observation apparently can be seen from the blue-shift in the Mn PL spectra after 10 days, presented in Fig. S10-12. For example, although at day 0, the Mn PL peak appears at ~620 nm for Mn-180 (7M) NPls, after 10 days, the Mn PL peak shifted to ~600 nm. Presumably, over time surface doping increases in the NPls from the surrounding Mn-OLA complex, leading to a more ligand field interaction (i.e. higher splitting of the 4 T1-6 A1 energy), causing the blue shift in the Mn PL. However, a detailed investigation is necessary to have a clear understanding of such doping mechanism and the resultant Mn PL tunability.

Estimation of the energy transfer efficiency and time.
To estimate the energy transfer efficiency, we model the bleaching dynamics (in Moreover, by recalling the PL lifetime measurement (Fig. 4, Fig. S13 and Table S2, S3), we found the undoped NPls prepared at a lower temperature (Un-100) has higher exciton lifetime than the one prepared at a higher temperature, Un-180 (4 ns vs. 3.1 ns).
This indicates the non-radiative exciton relaxation decreases in the Un-100 NPls due to reduced interaction with ligands (less surface passivated). Therefore, the exciton can transfer the energy more efficiently to the Mn-dopant in the Mn-100 NPls than the Mn-180 NPls. In short, the organic ligands act as a non-radiative center for exciton trapping and thereby reduced the energy transfer efficiency.


Ultra‐strong phosphorescence with 48% quantum yield from grinding treated thermal annealed carbon dots and boric acid composite

Metal‐free room‐temperature phosphorescence (RTP) materials are of great significance for many applications; however, they usually exhibit low efficiency and weak intensity. This article reports a new strategy for the preparation of a high‐efficiency and strong RTP materials from crystalline thermal‐annealed carbon dots (CDs) and boric acid (BA) composite (g‐t‐CD@BA) through grinding‐induced amorphous to crystallization transition. Amorphous thermal‐annealed CDs and BA composite (t‐CD@BA) is prepared following a thermal melting and super‐cooling route, where the CDs are fully dispersed in molten BA liquid and uniformly frozen in an amorphous thermal annealed BA matrix after super‐cooling to room temperature. Upon grinding treatment, the fracture and fragmentation caused by grinding promote the transformation of the high‐energy amorphous state to the lower energy crystalline counterparts. As a result, the CDs are uniformly in situ embedded in the BA crystal matrix. This method affords maximum uniform embedding of the CDs in the BA crystals, decreases nonradiative decay, and promotes intersystem crossing by restraining the free vibration of the CDs, thus producing strong RTP materials with the highest reported phosphorescence quantum yield (48%). Remarkably, RTP from g‐t‐CD@BA powder is strong enough to illuminate items with a delay time exceeding 9 s.

| INTRODUCTION
Room-temperature phosphorescence (RTP) materials with long emission lifetimes are highly desirable in a wide range of applications, such as anti-fake materials, sensors, and optoelectronic devices. [1][2][3][4] To date, most efficient RTP materials are limited to metal-doped inorganic complexes and organometallic compounds; 5 however, the high cost and cytotoxicity of these materials, owing to the presence of noble metals, limit their practical application. Recently, high-efficiency metal-free organic RTP materials have been realized by several strategies, [6][7][8][9][10] including crystal engineering, the heavy atom effect, and host-guest supramolecular systems. Most recently, Huang et al. reported an extra-high phosphorescence quantum yield (PQY, 46.1%) of a supramolecular crystal framework. 10 However, organic materials usually display poor photostability and require specific molecular designs to produce the desired photoluminescent properties, while challenges associated with a facile synthetic route remain.
Because the rigid crystal matrix can greatly reduce the proportion of nonradiative transitions, the embedding of phosphor into a crystalline material shows great potential in the preparation of high-efficiency RTP materials. 1,[33][34][35][36] Recently, RTP CD@crystal materials were developed by embedding CDs in crystal matrices under hydrothermal or solvothermal conditions. 25,37,38 However, CDs are generally prone to disperse in common solvents but tend to agglomerate on or among the crystals, thereby leading to weak intensity due to the low loading capacity in crystals. Moreover, for practical applications, high-performance optoelectronic devices require both a high RTP efficiency and strong RTP output. Thus, exploration of novel fabrication strategies to effectively disperse CDs in crystals to achieve highefficiency and strong RTP is highly desirable. With these facts in mind, we designed and developed a facile and novel strategy for the preparation of a high-efficiency and strong RTP materials from crystalline thermal-annealed carbon dot (CDs) and boric acid (BA) composite (g-t-CD@BA) through grinding induced amorphous to crystallization transition. The g-t-CD@BA displayed strong phosphorescence with an ultrahigh PQY of 48%, which, to the best of our knowledge, is the highest value reported for RTP CD-based materials to date. Notably, after switching off the 365 nm irradiation, the RTP of the g-t-CD@BA is strong enough to illuminate items with a delay exceeding 9 s and RTP luminance reaches a value of 590 lux at a delay of 1 s.

| RESULTS AND DISCUSSION
The CDs were synthesized by one-pot hydrothermal treatment of lycorine hydrochloride at 200°C for 2 h, and then composited with BA through thermal melting followed by super-cooling treatment to prepare amorphous CDs and BA composite (t-CD@BA). (Figure 1, detail in the Supporting Information Material). During this process, molten BA acts as a medium to allow the CDs to fully disperse into the matrix and is then uniformly frozen in the amorphous thermal annealed BA matrix after super-cooling to room temperature. Upon mechanical grinding, a sharply increased surface energy of the ground fine fragments drives the rearrangement of the contained disordered BA molecules to an ordered structure, resulting in BA crystallization. Notably, in this process, the CDs are in situ and homogeneously embedded in the crystalline BA matrix. This method affords maximum uniformly embedded CDs in the BA crystals. It also decreases nonradiative transition and increases ISC in the CDs by restraining the vibrational freedom of the CDs as a result of the rigid BA crystal matrix.
In the transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images (Figure 2A), the CDs show an average diameter of 5 ± 1.2 nm and a wellresolved lattice spacing of 0.21 nm, corresponding to the (100) facet of graphite carbon. 14 The chemical structure and photophysical properties of the CDs and t-CD@BA were investigated. The Fourier transform infrared (FTIR) spectra ( Figure 2B) reveal that the CDs exhibit characteristic absorption peaks at 3400, 1631, and 1617 cm −1 , assigned to the O─H, C═O, and C═C stretching vibrations, respectively. 27 After incorporating the CDs into the thermal annealed BA matrix, new absorption peaks can be observed at 941 cm −1 , corresponding to the C─B stretching vibration, 18,21,22 indicating BA molecules functionalized on the CDs after thermal treatment. In the XPS results, the high-resolution B 1 s spectrum of t-CD@BA ( Figure 2C) displays three bands centered at 194. 6, 193.6, and 192.7 eV, assigned to B─O, B 2 O 3 , and BCO 2 , respectively, 18 implying dehydration process occurs among CDs and BA molecules. These results indicate that the CDs were chemically bonded in the BA matrix.
In the photoluminescence (PL) spectra ( Figure 3A), the CD aqueous solution comprises a main emission peak at 430 nm, while the t-CD@BA displays two significant emission peaks approximately at 430 and 530 nm, respectively. Moreover, the emission peak at 530 nm in the PL spectrum of t-CD@BA almost coincides with its peak in the RTP spectrum. When t-CD@BA is fully dissolved in water, the peak at 530 nm nearly disappears in FL spectrum and no phosphorescent emission is observed ( Figure S1). It is well known that F I G U R E 1 Design strategy of ultra-strong CD@BA RTP material. Schematic illustration of the preparation procedure for the CDs, t-CD@BA and g-t-CD@BA. CD, carbon dot; RTP, room-temperature phosphorescence phosphorescence quenching usually occurs in the presence of water owing to the presence of dissolved oxygen as well as the occurrence of solvent-assisted relaxation. These results indicate that the peak at 530 nm in the form is mainly derived from phosphorescence. Thermal annealed BA sample exhibited negligible fluorescence and phosphorescence compared with that of t-CD@BA at room temperature ( Figure S2), indicating the RTP mainly comes from the chemically embedded CDs.
Interestingly, the t-CD@BA sample after grinding (g-t-CD@BA) displayed an unprecedented RTP enhancement phenomenon (~3.2-fold increase) ( Figure 3B). In phosphorescence decay curves ( Figure 3C and Table S1), enhanced average lifetime and strength of RTP were detected from g-t-CD@BA compared with that of t-CD@BA. The total QY and PQY of t-CD@BA were measured to be 19% and 15%, respectively. In contrast, the total QY and PQY of g-t-CD@BA significantly increased to 64% and 48%, respectively. The g-t-CD@BA not only exhibits a high RTP efficiency but also an extraordinarily strong RTP intensity. Remarkably, after switching off the 365 nm irradiation source (5 W), the intense green RTP from a plate (5 × 5 cm) filled with g-t-CD@BA can illuminate items for more than 9 s ( Figure 3D and Movie S1). The RTP luminance value of g-t-CD@BA can reach 590 lux at a delay of 1 s, which is exceedingly higher than the brightness (97 lux) of commercial mobile phone screens ( Figure 3E and Movie S2). For comparison, some typical luminance values in real environments are sunrise/sunset on a clear day (400 lux), office lighting (320-500 lux), and typical TV studio lighting (1000 lux). Interestingly, even after being excited by sunlight on a dark or cloudy day, the g-t-CD@BA still exhibited a bright green RTP, visible to the unaided eye, with a delay of 9 s ( Figure 3F and Movie S3), which, to the best of our knowledge, has never been reported. Phosphorescence excitation (PLE) spectra of the g-t-CD@BA indicate ultraviolet light in sunlight plays a major contribution to phosphorescence emission ( Figure S3). In addition, the phosphorescence of the g-t-CD@BA can last for about 13 s, visible to the unaided eye, after being excited by 365 nm light ( Figure S4).
To reveal the grinding-induced RTP enhancement phenomenon, we next investigated the changes between the structures of t-CD@BA and g-t-CD@BA. Under mechanical grinding, large lumps (>1 × 1 mm) of t-CD@BA are ground into g-t-CD@BA in fine particles in the size of 40-60 μm, as shown in Figure S5. The X-ray diffraction (XRD) spectrum ( Figure 4A) for the t-CD@BA presented broadened and weak peaks, indicative of an amorphous phase. [39][40][41] Following grinding, the spectrum of the g-t-CD@BA presented several sharp diffraction peaks which is similar to crystalline BA powder ( Figure S6), indicating a grinding-induced structural transition from an amorphous to a crystal phase. In the differential scanning calorimetry (DSC) curves on heating above melting point (Figure 4B), the t-CD@BA exhibited two weak endothermic peaks at 110°C and 152°C with total enthalpy of 333 J/g. On the other hand, for the g-t-CD@BA, the endothermic peaks became much stronger with total enthalpy of 1080 J/g, indicating that g-t-CD@BA possesses much lower energy than t-CD@BA. 40 This further confirms the grinding induced transition from the high-energy amorphous phase to a low-energy crystal phase. The formation of the amorphous t-CD@BA mainly occurs because the molten BA molecules are unable to rearrange into a crystalline lattice and are therefore "frozen" into a more disordered state during the rapid cooling process. Compared with the amorphous environment, a more rigid crystalline environment can effectively restrict the freedom of the embedded CDs by restraining the vibrations of their surface groups to reduce nonradiative relaxation and enhance the ISC process, leading to greatly enhanced RTP.
Based on the results above, it can be inferred the rapid change of the specific surface area of t-CD@BA from large lumps to fine particles, results in a sharply increased surface energy, which is not stable in room temperature. 41,42 Thus, a more stable crystal phase with lower energy is formed from amorphous phase of the thermal annealed BA matrix, resulting in the formation of crystalline g-t-CD@BA.
To clarify the superiority of our method, we next performed control experiments to compare as-prepared samples with those attained by traditional crystallization process. Thus, CDs and BA composites through evaporative crystallization (e-CD@BA) with different CDs loadings were prepared by low-temperature evaporation of mixture solutions of CDs and BA molecules, where CDs and BA were in the same proportions as in the t-CD@BA samples. The results clearly revealed that all the t-CD@BA samples were transparent without any apparent aggregation and delamination under daylight ( Figures 5A and S7). The PL and RTP images ( Figure 5A) reveal uniform luminescence for t-CD@BA and g-t-CD@BA, implying the homogeneous dispensation of the CDs into the thermal annealed BA matrices. In contrast, the CDs were severely agglomerated among the BA F I G U R E 4 XRD and DSC characterization of the CD@BA composites. (A) XRD spectra, (B) DSC curves of t-CD@BA and g-t-CD@BA on the heating run. BA, boric acid; CD, carbon dot; DSC, differential scanning calorimetry; XRD, X-ray diffraction crystals, even under low-loading conditions. Nonuniform PL and RTP from e-CD@BA and ground e-CD@BA (g-e-CD@BA) were observed at different CD concentrations ( Figures 5B and S8).
In the phosphorescence spectra ( Figures 5C and S9a), the phosphorescence emission intensity of g-t-CD@BA gradually increases with increasing CDs loading fraction, reaching the maximum value at a concentration of 4.5 mg/g. However, for g-e-CD@BA, the strongest phosphorescent emission was only obtained at a lower concentration of 0.3 mg/g, which is much lower than that for g-t-CD@BA, implying a low loading capacity of the CDs in evaporative crystallized BA matrix ( Figures 5C  and S9B). The absorbance spectrum of g-e-CD@BA at a concentration of 1.5 mg/g exhibits enhanced tails at the longer wavelength region compared with that of the g-t-CD@BA ( Figure 5D), indicating CDs aggregation among the evaporating formed BA crystals. During evaporative crystallization, the CDs are more prone to aggregate on or among the BA crystals, resulting in a weak On the other hand, for the g-t-CD@BA, maximum uniform embedding of the CDs in the BA samples is observed. Thus, g-t-CD@BA can still hold high optical quality, even under high-loading conditions. Based on the above results, we propose a grindinginduced crystallization strategy for the preparation of high-efficiency and strong RTP from CDs and BA composite according to the schematic illustration presented in Figure 6. First, amorphous t-CD@BA facilitates uniform embedding of the CDs into the thermal annealed BA matrix, which effectively avoids agglomeration of CDs. The higher energy amorphous t-CD@BA is not stable, which tends to transform to lower energy crystalline state upon external mechanical stimulus. After grinding, the sharply increased surface energy of the ground fine fragments drives the rearrangement of the contained disordered BA molecules to an ordered structure, thus leading to instant transition from amorphous to crystal phase, as demonstrated by XRD results. Thus, the CDs are in situ and homogeneously embedded in the crystalline BA matrix. Compared with the amorphous environment, a more rigid crystalline environment can effectively reduce nonradiative transitions and enhance the ISC process by restraining the vibrations of CDs, thus leading to greatly enhanced RTP. For the evaporative crystallization method, the CDs are generally more prone to aggregate on or among the developed BA crystals, thus leading to low RTP efficiency and weak emission.

| CONCLUSION
In summary, we have successfully exploited a new grinding-induced amorphous-to-crystalline transition method for the preparation of high-efficiency RTP materials. The as-prepared g-t-CD@BA exhibits both a high RTP efficiency (PQY of 48%) and strong RTP emission. Remarkable, RTP from g-t-CD@BA is strong enough to illuminate items with a visible time exceeding 9 s to the unaided eye, and RTP luminance value of 590 lux at a delay of 1 s. The excellent RTP properties were mainly attributed to the proposed newly fashioned grindinginduced phase transformation method. In this method, thermal annealed amorphous BA facilitates maximum uniformly embedding of the CDs into the matrix. Moreover, the grinding-induced instant crystallization allows the CDs to be in situ and homogeneously embedded into the crystal BA matrix, greatly surpassing the nonradiative transition and promoting the ISC of CDs. This study provides a practical synthetic strategy for the large-scale preparation of high-efficiency RTP materials for various applications.
F I G U R E 6 Schematic of the proposed phosphorescence mechanism of CD@BA composites. Possible photoluminescence emission processes and energy-level diagram of t-CD@BA, g-t-CD@BA and g-e-CD@BA. (Abs., absorption; Fluor., fluorescence; Vib., vibration)


s2fieldsofstudy:
Chemistry
Materials Science

text:
One-Pot Synthesis of Pt Nanobowls Assembled from Ultrafine Nanoparticles for Methanol Oxidation Reaction

Simultaneously engineering a bowl-like and ultrafine nano-size structure offers an attractive route to not only increase the utilization efficiency of noble metals, the specific surface areas and the availability of active sites, but also boost the structural robustness and long-term stability. However, a great challenge remains in terms of the methods of synthesis. Herein, we report a facile one-pot hydrothermal method for the preparation of hollow porous Pt nanobowls (NBs) assembled from ultrafine particles. N,N′-methylenebisacrylamide (MBAA) acts as a structure-directing agent that forms a self-template with Pt ions and drives the nucleation and assembly of Pt metals, resulting in the fabrication of Pt NBs from ultrafine particles. By virtue of their unique structure and morphology, the optimized Pt NBs exhibited enhanced electrocatalytic methanol oxidation reaction (MOR) activity with 3.1-fold greater mass activity and 2.6-fold greater specific activities compared with those of commercial Pt black catalysts, as well as excellent stability and anti-poisoning ability.

Introduction
Facing a series of problems caused by the consumption of fossil fuels and environmental pollution, there is an urgent need to develop sustainable and clean energy conversion devices as substitutes for conventional internal combustion engines [1][2][3][4]. In this context, direct methanol fuel cells (DMFCs) have rapidly attracted increasing attention among the various renewable energy devices due to their high energy conversion efficiency, high power density, ready availability for transportation and storage, and zero carbon footprint [5][6][7][8]. However, the sluggish kinetics of anodic MORs has severely impeded the progress and application of DMFCs [7,9,10]. Hitherto, Pt nanomaterials are still regarded as the most efficient and extensively used MOR catalysts due to their extraordinary affinity for methanol molecules and minimal activation barriers [11][12][13][14][15]. Although many merits of MORs have been recognized, the scarcity, insufficient durability and CO poisoning of commercial Pt electrocatalysts are still major challenges affecting the future development of DMFCs [16][17][18]. Therefore, it remains desirable to develop high-efficiency Pt electrocatalysts with minimal Pt content as well as promising activity and durability towards MORs [19][20][21].
One effective strategy is to tune the size and morphology of Pt catalysts, because the electrochemical performance of catalysts is also highly dependent on these in addition to the components [22][23][24]. As shown by many reported cases, the size effect of noble metals plays a key role in catalyzing a series of important chemical reactions [25][26][27][28]. Notably, reducing the particle size can not only maximize the surface exposure and improve the utilization efficiency of noble metals' atoms, but can also simultaneously affect the electronic structure of the active metals [29][30][31][32][33][34][35]. Fan et al., for example, studied the catalytic activity of gold nanoparticles with different sizes. The experimental results showed that the catalytic performance decreased with an increase in the nanoparticles' size [36]. Yang et al. investigated the effect of the size of PdCu nanocatalysts on the ORR activity. The ORR mass activity of PdCu nanoparticles increased from 0.135 A·mg −1 to 0.307 A·mg −1 as the size decreased from 9.3 nm to 5.8 nm. Density functional theory (DFT) studies have revealed that the edges and corners of metal nanoparticles increase with decreasing size, leading to enhanced ORR catalytic activity [37]. However, controlling the size of ultrafine Pt nanoparticles (under 5 nm) remains a challenge due to their high specific surface energy, which is prone to aggregation and migration during synthesis and electrocatalysis, thus greatly diminishing their catalytic performance [38,39]. The common strategies of integrating the supports to control the nanoparticle size suffer from multiple synthesis steps, non-uniform particle sizes and reduced exposure of the catalyst's active sites [40,41]. To overcome these difficulties, the uniform assembly of ultrafine nanoparticles into a hollow and porous bowllike structure is a powerful and unusual strategy, which not only maintains robust stability to prevent the aggregation and migration of ultrafine nanoparticles without introducing supports, but also inherits the high catalytic activity of ultrafine nanoparticles and ensures the maximum retention of active sites [42][43][44][45][46]. Furthermore, the unique hollow bowls are beneficial to provide more open pores and fewer diffusion blockages, resulting in the rapid and efficient entry of reactants and electrolytes into the void space, accelerating the reaction kinetics [47][48][49][50]. However, similar to ultrafine nanoparticles, the synthesis of hollow bowl-like structures still faces great challenges. The template method and galvanic displacement reactions are widely used to synthesize hollow porous structures, which often encounter setbacks such as complicated operation and multiple steps [51][52][53]. Therefore, it is of great significance to develop a simple and effective synthesis strategy that can integrate the merits of ultrafine nanoparticles and hollow porous nanobowls.
Inspired by the approaches above, herein, we developed a simple and ingenious onepot hydrothermal approach to synthesize Pt NBs assembled from ultrafine nanoparticles by utilizing MBAA. MBAA possesses two amide groups ( Figure S1) that can efficiently complex Pt ions, inducing the reduction of Pt ions and the formation of bowl-like structures. Such unique hollow and porous nanobowls endow the catalysts with large surface areas, rich active sites and rapid permeability. As electrocatalysts for the MOR, the as-prepared Pt NBs exhibited superior specific activity (2.8 mA cm −2 ) and mass activity (507.8 mA mg −1 ), as well as enhanced stability and CO ads poisoning tolerance compared with commercial Pt black catalysts.

Reagents and Chemicals
MBAA was purchased from Alfa Aesar Co., Ltd., Heysham, UK. Hexachloroplatinic (IV) acid hexahydrate (H 2 PtCl 6 · 6H 2 O) was purchased from Shanghai Dibai Biotechnology Co., Ltd, Shanghai, China. Formaldehyde (HCHO) and methanol (CH 3 OH) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. The Pt black catalyst was ordered from Johnson Matthey & Co., Ltd., USA. All reagents were of analytical grade and were used without further purification.

Methods
To synthesize the Pt NBs, 1 mL of a 0.05 M H 2 PtCl 6 solution and 50 mg MBAA were added into 17.0 mL deionized water with continuous ultrasonic stirring for 1 h at room temperature. Next, 1 mL of a HCHO solution was added into this solution. After ultrasonic treatment for 5 min, the resulting solution was transferred to a 25 mL Teflon-lined stainless steel autoclave and heated at 160 • C for 6 h. After being cooled to room temperature, the final product was collected by centrifugation at 15,000 rpm for 5 min and further purified by washing three times with distilled water and ethanol.

Characterization
Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images of the Pt NBs were acquired on Talos G2 F20 and JEOL JEM-2100F instruments, manipulated with an accelerating voltage of 200 kV. Scanning electron microscopy (SEM) studies were performed using a Hitachi S4800 system. XRD analyses were performed on a Model D/max-rC X-ray diffractometer using Kα radiation (λ = 0.15406 nm). XPS measurements were conducted on a Thermo VG Scientific ESCALAB 250 spectrometer with Al Kα radiation. The binding energy was calibrated by means of the C 1s binding energy at 284.6 eV.

Electrochemical Measurements
Electrochemical measurements were performed on a CHI 760E CH analyzer (Shanghai, China, Chenhua Co., Ltd.) with a conventional three-electrode system. The glassy carbon electrode loaded with a catalyst was used as the working electrode, with the graphite rod as the counter-electrode and a saturated calomel electrode (SCE) as the reference electrode for the MOR tests. The catalyst ink was prepared by ultrasonically dispersing a mixture of 2.0 mg of the catalyst and 1 mL of deionized water. Next, a 6 µL drop of the suspension was coated on a glassy carbon electrode with a diameter of 3 mm. After drying, 3 µL of a Nafion solution (5 wt.%) was added onto the surface of the modified electrode and dried again naturally at room temperature. Electrochemical measurements were performed in a 0.5 M N 2 -saturated H 2 SO 4 solution with or without 1.0 M CH 3 OH at a scan rate of 50 mV s -1 . Chronoamperometry curves were obtained in a N 2 -saturated 0.5 M H 2 SO 4 + 1 M CH 3 OH mixed solution for 85,000 s at 0.6 V. Figure 1a illustrates the one-pot synthesis of Pt NBs. In a typical process, the Pt NBs were successfully constructed with the assistance of MBAA, using H 2 PtCl 6 as the metal precursor and HCHO as the reducing agent. The crystalline nature of the Pt NBs was confirmed by powder X-ray diffraction (XRD). As obviously shown in Figure 1b Figure 1c shows that the deconvoluted Pt 4f spectrum of Pt NBs was composed of Pt 0 and Pt 2+ species. The peaks located at 70.8 and 74.1 eV were attributed to Pt 4f 7/2 and Pt 4f 5/2 of Pt 0 species, respectively, whereas the other low peaks at 71.8 and 75.1 eV can be attributed to Pt 2+ species [54]. Through integration of the peak areas, 72.3% of the Pt elements were reduced to metallic Pt 0 , verifying the effective reduction of Pt 4+ . Compared with that of standard metallic Pt (4f 7/2 : 70.90 eV), the negative shift of about 0.1 eV was probably due to the surfacemodifying effect of MBAA on Pt [55]. Moreover, the modification of a small amount of MBAA on Pt was further confirmed by the N 1s spectrum ( Figure S2).

Results and Discussion
The morphology and structure of the resulting Pt NBs were initially characterized by various electron microscopy techniques. As revealed in the SEM images ( Figure 2a and Figure S3), the obtained Pt NBs exhibited a uniformly dispersed bowl-like structure with an almost 100% yield. TEM images at different magnifications further validated the hollow bowl-like nature, with an average diameter of 300 nm (Figure 2b,c). Unlike the traditional single-layer bowl-like structure, the Pt NBs obtained here possessed double-layered shells, similar to a concave hollow spherical structure. Notably, further magnification of the TEM images (Figure 2d and Figure S4) revealed that the obtained concave bowl-like structure was constructed by interconnecting and assembled Pt nanoparticles with a size of about 3.45 nm. Figure 2e presents the HRTEM image of the Pt NBs. The adjacent lattice fringes of the majority of the exposed facets were 0.225 nm, perfectly corresponding to the (111) lattice spacing of fcc Pt. In the enlarged HRTEM image, a large number of lattice defects such as distortions and twin boundaries can clearly be observed on the surface of the Pt NBs, as indicated by the yellow markers ( Figure S5). Moreover, the surface of the nanobowl was rough and porous, favorably reducing the mass transfer resistance and improving the reaction kinetics in the electrocatalytic process (red marks in Figure S5) [47,56]. Meanwhile, the hollow and porous bowl-like structure with abundant surface defects could provide large electrocatalytic surface areas and accessible active sites, thus facilitating improvements in the electrocatalytic performance. The clear bowl-like structure was also confirmed by the scanning TEM (STEM) images (Figure 2f,g). As demonstrated in Figure 2f, the dark centers and bright edges can clearly be observed, which are distinct features of nanobowls. The bowl-like structure was further verified by the energy-dispersive X-ray (EDX) line scanning spectra, according to the weaker element signals in the hollow center (inset in Figure 2f). Combined with the EDX element mapping images in Figure 2h-j, the uniform distribution of Pt and a small number of N elements were detected on the surface of the Pt NBs. The modification of a small number of N elements on the surface may induce changes in the electronic structure of Pt, which is beneficial for relieving the adsorption of intermediates, thereby boosting the electrocatalytic reaction performance. To understand the role of MBAA in adjusting the morphology, comparative experiments with different amounts of MBAA were carried out. In the absence of MBAA ( Figure S6a), the products were small agglomerated nanoparticles with no specific morphology. When 5 mg of MBAA was added, nanospheres with a size of about 70 nm were obtained ( Figure S6b). As the amount of MBAA increased to 10 mg ( Figure S6c), the centers of the nanospheres began to form hollows, accompanied by some by-products. When the amount of MBAA reached 15 mg (Figure S6d), the hollow nanospheres gradually became concave and the by-products decreased significantly. As the amount of MBAA continued to increase to 30 mg ( Figure S6e), apple-like structures were formed. The detailed experimental results show that the concave degree of the product gradually increased with the increase in the amount of MBAA. When 50 mg of MBAA was added ( Figure S6f), uniform hollow porous nanobowls were finally obtained. These results suggest that MBAA plays an indispensable role in forming the hollow bowl-like morphology of the catalyst. Reasonably, MBAA, as a strong ligand with two special amide groups, can effectively complex with Pt ions. As confirmed by the UV-Vis spectra ( Figure S7a), the characteristic absorption peak of the H 2 PtCl 6 solution at 261 nm disappeared after mixing H 2 PtCl 6 and MBAA solution, fully confirming the complexation of the two solutions. The complexes thus formed can effectively reduce the reduction rate of Pt ions, thereby reducing the rate of self-nucleation and growth, and regulating the final morphology of the product [57,58]. In addition, FT-IR spectroscopy was performed on the MBAA and the final Pt NBs ( Figure S7b). The FT-IR spectra of the MBAA and the final Pt NBs showed that there was a blue shift in the amine group on the surface of the Pt NBs, showing that there was interaction between Pt and the amine group of MBAA. The modification of a small amount of MBAA on Pt is conducive to changing the electronic structure of Pt and easing the adsorption of intermediates, thus improving the electrocatalytic performance [55]. To further clarify the morphological evolution of Pt NBs, the intermediates collected at different reaction times were analyzed using the controlled variable method. At the initial reaction time, the products were obtained as yellow powder. Additionally, the corresponding TEM images (Figure 3a,b) showed that the products were uniformly dispersed solid nanospheres with a smooth surface and an average particle size of 500 nm. However, the corresponding XRD pattern ( Figure S8a) indicated that there were no characteristic diffraction peaks of Pt. This result revealed that no metal Pt was reduced and the nanospherical products thus obtained were complexes formed by MBAA and Pt ions at high temperature and high pressure. With the prolongation of the reaction time to 2 h, ultrafine nanoparticles gradually grew on the surface of the nanospheres (Figure 3c,d), which were confirmed to be Pt nanoparticles by XRD ( Figure S8b). In addition, the nanospheres changed from solid to hollow, with a large amount of by-product, which could be attributed to the continuous diffusion and reduction of Pt ions on the surface of the nanospheres (Figure 3e,f). As the reaction time increased to 4 h, due to the further diffusion of and reduction in platinum ions, the number of complexes as supporting spheres gradually decreased, resulting in the surface depressions of hollow nanospheres. Moreover, with the further diffusion of and complete reduction in Pt ions, the concavity of the hollow nanospheres was further deepened. Finally, hollow and porous bowl-like Pt NBs assembled from ultrafine nanoparticles were obtained when the hydrothermal reaction time reached 6 h (Figure 3g,h). Figure 3i briefly illustrates the structural evolution of the Pt NBs. Unlike the traditional hard template method, using MBAA as the morphology-directing agent to prepare the hollow and porous bowl-like structure not only involves fewer steps and produces less waste, but is also cost-effective. The synthesis involves the complexation of MBAA and Pt ions, the soft template effects of the complex and the diffusion of Pt ions, which will provide new ideas for the formation of bowl-shaped Pt nanomaterials. Notably, bowl-shaped metals or nanomaterials have exhibited great potential in catalysis, energy storage and biomedical applications [52,59]. Inspired by their unique structural characteristics, the catalytic performance of Pt NBs and commercial Pt black were evaluated for MORs under acidic conditions. Before the electrochemical test, the prepared Pt NB catalyst was immersed in acetic acid for 12 h to remove excess MBAA on the surface. First, cyclic voltammetry (CV) measurements were recorded in a 0.5 M H 2 SO 4 solution at a scan rate of 50 mV s −1 . The electrochemically active areas (ECSAs) of the catalysts were calculated by integrating the charge in the hydrogen adsorption-desorption region of the cyclic voltammetry curves thus obtained (Figure 4a). The ECSAs of hollow Pt NBs and commercial Pt black were measured to be 18.2 and 12.5 m 2 g −1 , respectively. Due to their larger ECSAs, the Pt NBs were expected to be outstanding MOR catalysts. Therefore, the MOR electrocatalytic activity of the Pt NBs and Pt black catalysts were evaluated by CV curves obtained in a 0.5 M H 2 SO 4 + 1.0 M CH 3 OH solution. As indicated in Figure 4b, the mass activity of the Pt NB catalyst reached 507.8 mA mg −1 , with a 2.1-fold improvement compared with the commercial Pt black (242.7 mA mg −1 ). A similar trend also emerged in terms of the specific activity. As shown in Figure 4c, the specific activity of the Pt NBs was calculated to be 2.8 mA cm −2 , which was 1.4 times higher than that of commercial Pt black (1.9 mA cm −2 ). Figure 4d exhibits the corresponding Tafel curves derived from Figure 4c. The Tafel plots maintained a linear relationship in the low potential region. Additionally, the Pt NBs displayed a higher output current density than the commercial Pt black at the same potential, indicating a faster kinetic rate for methanol electrocatalysis (Figure 4d). Furthermore, the lower Tafel slope of the Pt NBs indicated that there was less coverage of CO or intermediates in the Tafel region compared with commercial Pt black. Electrocatalytic stability and CO poisoning tolerance are two important indicators for evaluating the performance of the MOR. Chronoamperometry was first used to assess the catalytic stability of Pt NBs and commercial Pt black in an N 2 -saturated 0.5 M H 2 SO 4 + 1 M CH 3 OH solution at a potential of 0.6 V. As shown in Figure S9, the current density of the Pt NBs was consistently higher than that of the commercial Pt black after 85,000 s, manifesting a more robust MOR stability. In addition, the morphology of the recovered Pt NBs retained the whole bowl-shaped structure without significant changes after the stability test. (Figure 5a). The excellent electrocatalytic stability of the Pt NBs resulted from its hollow porous structure, which effectively inhibited the possible aggregation, dissolution and Ostwald maturation of catalysts. The CO poisoning resistance of Pt NBs towards the MOR was investigated by CO stripping voltammetry (Figure 5b). Compared with those of commercial Pt black, the onset and peak oxidation potential of CO on the Pt NBs were negatively shifted by 140 and 110 mV, respectively, indicating stronger resistance to CO poisoning during the MOR electrocatalysis process. On the basis of the experimental results and analysis above, we can confirm that Pt NBs are excellent and stable catalysts for anode MOR of DMFCs. This can be attributed to the following aspects: (i) the hollow porous nanobowls and ultrafine nanoparticles can provide large specific surface areas and abundant accessible active sites, which are conducive to the rapid transport of mass and electrons, in favor of improvements in the MOR's activity; (ii) the ultrafine nano-size and the surface modification of MBAA can tune the electronic structure of Pt and facilitate the desorption of intermediates and toxic species, thereby enhancing the anti-poisoning ability and the reaction kinetics of the catalysts; (iii) the self-supporting property of the hollow bowl-like structure can effectively prevent the agglomeration, leaching and migration of the catalyst, firmly maintaining the whole structure during the electrocatalytic process.

Conclusions
In summary, we have successfully synthesized hollow porous Pt NBs composed of ultrafine nanoparticles (about 3 nm) via an easy one-pot hydrothermal method of synthesis. A series of experimental studies indicated that MBAA plays a decisive role in tuning the bowl-like structure. The successful integration of the hollow bowl-like structure and ultrafine nanoparticles provided large active areas, accessible active sites and rapid mass transfer channels for the Pt NBs catalyst. In virtue of their favorable morphology and electronic structure, the optimized Pt NBs exhibited remarkably improved electrocatalytic MOR activity, stability and anti-poisoning ability in an acidic medium. This study has presented an efficient method for and furthered our understanding of the formation of bowl-shaped nanomaterials.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nano12193471/s1. Figure S1: Molecular structure of MBAA. Figure S2. High-resolution N 1 s XPS spectrum of Pt NBs. Figure S3: SEM images of Pt NBs at different magnifications. Figure S4: HREM image of Pt NBs. Figure S5: HREM image of Pt NBs.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.


s2fieldsofstudy: Chemistry Biology

text:
Discovery of a fragment hit compound targeting D-Ala:D-Ala ligase of bacterial peptidoglycan biosynthesis

Abstract Bacterial resistance is an increasing threat to healthcare systems, highlighting the need for discovering new antibacterial agents. An established technique, fragment-based drug discovery, was used to target a bacterial enzyme Ddl involved in the biosynthesis of peptidoglycan. We assembled general and focused fragment libraries that were screened in a biochemical inhibition assay. Screening revealed a new fragment-hit inhibitor of DdlB with a Ki value of 20.7 ± 4.5 µM. Binding to the enzyme was confirmed by an orthogonal biophysical method, surface plasmon resonance, making the hit a promising starting point for fragment development.

Introduction
Infections caused by antibiotic-resistant bacteria pose a serious challenge to healthcare systems worldwide. The increasing resistance of gram-positive and gram-negative pathogens causing infections in hospitals and in the general population and the worldwide spread of antibiotic-resistant 'superbugs' represent a major global health problem 1 . Reportedly, 25,000 Europeans die each year as a direct result of infections with multidrug-resistant strains of pathogenic bacteria, with an estimated economic impact of 1.5e billion per year 2 . However, the pipeline for new drugs is small because major pharmaceutical companies have largely abandoned antibiotic research, despite the urgent need for new drugs 3 . The fact that few new antimicrobials are available and multidrug resistance is becoming more common means that we need to increase our efforts in finding new antimicrobials 4 . It is imperative that we continue to search for new antibacterial agents by using innovative screening methods for carefully selected protein targets and by conducting rational drug design using the advances offered by protein crystal structures.
Peptidoglycan is a macromolecule essential for bacterial survival and is found only in the bacterial cell wall. Therefore, enzymes involved in the peptidoglycan biosynthetic pathway represent potential targets for the discovery of new antimicrobial agents 5,6 . Among the intracellular enzymes involved in peptidoglycan biosynthesis, only two enzymes have been validated as antibacterial targets by inhibitors that are in clinical use: UDP-N-acetylglucosamine-enolpyruvyl transferase (MurA, EC 2.5.1.7) is validated by fosfomycin, which is used to treat urinary tract infections 7 , and D-alanine:D-alanine ligase (Ddl, EC 6.3.2.4) is validated by cycloserine (Figure 1(B)), which is a second-line drug for the treatment of tuberculosis 8 .
Ddl is an ATP-dependent bacterial enzyme that catalyses the ligation of two D-alanines to a D-alanyl-D-alanine product ( Figure  1(A)), which in the next step is incorporated into the final intracellular peptidoglycan precursor UDP-N-acetylmuramoyl pentapeptide 5 . Subsequently, this D-alanyl-D-alanine terminus is involved in transpeptidation, the cross-linking of the growing peptidoglycan chains 9 . In Escherichia coli, there are two isozymes of Ddl, DdlA and DdlB, which have similar catalytic efficiencies and substrate recognition properties, as well as similar sensitivity to inhibitors 10 . We focussed on the search for inhibitors of DdlB because it is the best studied and the crystal structures are available 11 . Although many Ddl inhibitors have been discovered in the past two decades, mainly by various screening campaigns or by classical medicinal chemistry approaches (for a comprehensive review, see ref. 12 ), these inhibitors usually had only weak or no antibacterial activity. The most promising recent inhibitors appear to be thiosemicarbazide 1 and thiosemicarbazone 2 (Figure 1(B)), which had promising antibacterial activity and have been shown to target Ddl in bacteria 13,14 .
One of the innovative and underutilised approaches to target Ddl is fragment-based drug discovery (FBDD). This approach is increasingly being used in the pharmaceutical industry and academia to reduce attrition and provide leads for previously inaccessible biological targets. FBDD identifies low molecular weight ligands ($150 Da) that bind to biologically important macromolecules. The three-dimensional experimental-binding mode of these fragments is determined by X-ray crystallography or NMR spectroscopy and is used to facilitate their optimisation into potent lead compounds with drug-like properties by known methods such as fragment growing and fragment linking. Compared with high-throughput screening, the fragment-based approach requires a smaller number of compounds to be screened and provides more efficient and fruitful optimisation campaigns 15,16 . In this manuscript, we describe the construction of a new library of fragments and its screening on DdlB from E. coli to discover new fragment-hit inhibitors.

Results and discussion
The definition of fragments in this study was based on physicochemical properties, the most important criterion being a molecular weight not exceeding 300 Da. Half of the fragment library was designed for a general purpose and will be screened on other targets as well. It contained diverse fragments (purchased or available in-house) without reactive functional groups. The second half consisted of subsets specifically designed to target DdlB. Since the enzyme is a dinuclear Mg 2þ -dependent metalloenzyme, fragments capable of chelating metal ions could potentially bind to the active site via coordination interactions with Mg 2þ ions. A subset of chelating fragments was designed using substructure filters for chelating groups (Supplementary Table S2) 17,18 . DdlB is also ATPdependent, thus we designed a subset of fragments containing phosphate bioisosteres (Supplementary Table S3) aiming to bind into the phosphate-binding site pockets normally occupied by ATP 19 . Next, we designed a subset of cycloserine analogues that have similar substructures to an approved drug cycloserine and could potentially bind to the D-Ala-binding site (Supplementary Table S4). The final subset was designed by selecting the highest scoring hits obtained by docking a virtual library of available fragments to ATP-and D-Ala-binding sites. In total, the fragment library contained 943 compounds (Table 1) distributed across a range of physicochemical properties (Supplementary Figure S1).
The fragment library was assayed in a DdlB inhibition assay, where fragments were tested for their ability to inhibit the D-Alaadding activity of DdlB ligase. The orthophosphate formed during the enzymatic reaction was measured spectrophotometrically using malachite green reagent. The tested fragment concentrations varied from 1.5 to 5 mM. The criteria for classifying a fragment as a hit were: <50% residual DdlB activity and solubility (low background absorbance of the compound at 650 nm). The results are provided in a Supplementary excel file. We re-purchased six hit compounds that matched the criteria to determine the inhibition of DdlB from freshly prepared compound solutions (Table 2). However, only two hits showed sufficient inhibition for IC 50 determination (3 and 4).
To pursue only compounds with tractable mechanism of action, extensive profiling of fragment hits was performed. First, deselection was performed with a MurA inhibition assay using the same assay technology to find compounds that interfered with the malachite green assay system. Second, the fragment hits were tested in a phosphate-binding assay that omitted the enzyme and again used the same assay technology. Compounds 4 and 5 were found to interfere with the assay by inhibiting MurA, DdlB, and binding phosphate ( Table 2). Redox activity assays were performed to find compounds that interfered with the assay or had undesirable mechanisms of bioreactivity leading to false positive inhibitions. The horseradish peroxidase-phenol red (HRP-PR) assay was used to detect H 2 O 2 generated by redox cycling compounds in the presence or absence of DTT. A fluorescent probe, 2 0 ,7 0dichlorodihydrofluorescein diacetate (H 2 DCFDA), was used to detect compounds generating reactive oxygen species in the presence or absence of a reducing agent TCEP. The resazurin assay was used to detect redox active compounds that catalyse the conversion of resazurin to resorufin by the formation of free radicals in the presence of the reducing agent DTT. Compounds 4-6 and 8 were found to be active in at least one of the redox activity assays ( Table 2, Table S5). Because our goal was to discover noncovalently binding inhibitors of DdlB, the fragment hits were assayed for their potential reactivity with a nucleophilic thiol surrogate, reduced 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Fragment 4 was found to be reactive in this assay (Table 2 and Supplementary Figure S2).  Based on the fragment hit profiling results, compounds with at least two red flags were excluded. For further evaluation, we selected fragment hit 3 without red flags and with an IC 50 value of 168 lM (Figure 2(A) and Table 2), which is in the same range as that of the approved drug cycloserine (IC 50 ¼ 108 lM). Compound 3 was part of a Docked fragments to DdlB subset of the fragment library. In particular, docking to the ATP-binding site was used in the selection of this compound as part of the fragment library design. Structurally, 3 is a pyrazolopyrimidinecarboxylic acid with a molecular weight of 253 Da.  In an attempt to investigate the structure-activity relationship, 15 analogues of 3 were purchased (analogue-by-catalogue) ( Table  3). Compounds 9-14 differ by the substituents on the phenyl ring, while other heterocycles were explored with compounds 15-18. The carboxylic group was switched from position 3 to position 2 for compounds 18 and 19. Compounds 20-23 contain isosteric replacements for the carboxylic group. None of the assayed analogues inhibited DdlB in an inhibition assay. However, because they are fragment-sized compounds (molecular weight between 234 and 322 Da), even small changes may contribute to loss of inhibitory potency 20 .
Binding of 3 to DdlB was confirmed by an orthogonal biophysical assay, surface plasmon resonance (SPR). The protein was covalently attached to the surface of the CM5 chip ($7700 response units), and the ligand was titrated across the surface (Figure 2(B)). The apparent equilibrium dissociation constant (K D ¼ 3.2 mM) was estimated from the steady-state binding levels ( Figure 2(C)). Steady-state kinetics were performed to gain mechanistic insight into the inhibition of compound 3 (Supplementary Figure S3 and Table S6). The resulting best model showed that 3 is a competitive inhibitor with respect to ATP with a Ki value of 20.7 ± 4.5 mM. These data are in agreement with the docking results obtained during the design of fragment library, where 3 was identified as a potential ATP competitive inhibitor.
Compound 3 was then additionally docked using extra-precision Glide (Glide XP) 21 and also redocked using QM-Polarized Ligand Docking (QPLD) protocol 22 that generates partial charges on the ligand atoms by quantum mechanical calculations on the ligand in the field of the receptor. This accounts for the polarisation of the charges on the ligand by the receptor environment, and redocking with these new charges can lead to improved docking accuracy. The QPLD pose showed a difference of 2.54 Å from the original pose, mainly due to the m-tolyl ring flip ( Figure  2(E)). Based on the putative binding mode, the carboxylic acid mimics the phosphate groups of ATP that coordinate Mg 2þ ions ( Figure 2(D,E)). As shown in Table 4, the electrostatic term (XP Electro) from Coulombic and metal interactions contributes the most, followed by XP Zpotr, which denotes a reward for ligand atoms placed in a favourable electrostatic environment of the protein, and XP PhobEn, a reward for hydrophobic enclosure. Thus, the appropriately positioned carboxylate group is mainly responsible for successful binding.  To confirm the relevance of the predicted docking pose, a 100 ns molecular dynamics (MD) simulation on the 3 binding pose was run. The pose was fairly stable during the simulation time (Supplementary Figure S4), with the largest fluctuations resulting from the flipping of the m-tolyl ring. The metal coordinating and ionic interactions with both of the active site Mg 2þ ions through the carboxylate moiety and one pyrimidino nitrogen anchored 3 in the active site (Supplementary Figure S5). Additionally, a cationp interaction of the m-tolyl ring with Lys144 occurred quite frequently. Overall, in the absence of an experimental crystal structure, these results provide strong evidence for the proposed binding mode of compound 3 in the ATP-binding site.
The antibacterial activities of 3 were determined according to the European Committee for Antibacterial Susceptibility Testing recommendations and Clinical Laboratory Standards Institute protocol 23 . Compound was tested against reference strains of E. coli and Staphylococcus aureus and against two mutant E. coli strains. E. coli D22 bears a mutation in the LpxC gene that increases membrane permeability, whereas E. coli N43 is an AcrA knockout strain (knockout of the cell membrane pump). The 3 showed no antibacterial activity against both E. coli and S. aureus (Table S7). The most plausible reason is the low on target activity of the hit fragment, however, also the poor penetration into the bacterial cell or efflux pumps effect cannot be ruled out. Although the lack of antibacterial activity is a limitation, hit compound 3 represents a fragment-sized starting point for the development of new compounds with increased DdlB inhibitory potency as well as antibacterial activity.

Conclusions
Here, we describe the construction of a fragment library containing 943 fragments to discover novel inhibitors of a bacterial enzyme DdlB involved in intracellular steps of peptidoglycan biosynthesis. FBDD is an innovative and underutilised approach to target DdlB. The library contained diverse fragments suitable for screening on different targets, as well as fragments specifically designed to target DdlB. Library screening using a biochemical assay was followed by profiling of the fragment hits. Compounds that interfered with the assay, redox active compounds, and reactive compounds were flagged. The fragment hit 3 without flags and with an IC 50 value of 168 lM was selected for further studies. We confirmed binding of 3 to DdlB with SPR analysis. Furthermore, steady-state kinetics showed 3 to be a competitive inhibitor with respect to ATP with a Ki value of 20.7 ± 4.5 mM. An analogue-by-catalogue campaign did not reveal more potent compounds, making fragment hit 3 a starting point for further synthetic optimisation to develop new antibacterial agents.

Fragment library preparation
The in silico part of the fragment library preparation was performed using the KNIME analytics platform 24 . SMARTSview server was used to visualise the SMARTS patterns 25 . For fragment library preparation, we selected compounds from more than 4500 inhouse chemicals and more than 1.57 million compounds available for cherry-picking from ChemDiv (the database was downloaded on 05 October 2020). First, the RDKit Descriptor Calculation node 26 was used to calculate the molecular descriptors for each compound in the library. Then, the compounds were filtered using the following criteria to retain only fragments: molecular weight between 80 and 300, no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, and estimated logP value no greater than 4. The Speedy SMILES Organic Subset Molecules Splitter node was used to remove compounds with non-organic atoms other than C, N, O, S, P, B, F, Cl, Br, I, and H. To remove reactive and unwanted functional groups, a set of SMARTS filters (Supplementary Table S1) were applied using the SMARTS Query node 27 . Finally, RDKit Molecule Catalogue Filter node was used to remove PAINS compounds known to cause interference in assay systems 28 . The resulting virtual fragment library of 1087 in-house and 91,379 commercially available compounds was then used to design six subsets.
The selected commercially available fragments were obtained at 1.0-1.2 mg on 96-well polypropylene plates. To each well, 100 lL of dimethyl sulfoxide (DMSO) was added, and the plates were covered with an aluminium seal, and sonicated to prepare 26-125 mM stock solutions, which were used for screening in a DdlB inhibition assay.
In-house diverse fragments Additional physicochemical filters were applied to the in-house available fragments, namely no more than 5 hydrogen bond acceptors, no more than 3 rotatable bonds, and a polar surface area greater than 0 and no greater than 80, resulting in a library of 887 in-house available fragments. Then, 400 diverse fragments were selected using the RDKit Diversity Picker node. The node uses a fast MaxMin algorithm to select diverse compounds based on the Tanimoto distance between the Daylight-like topological fingerprints. The diverse fragments were then manually inspected and a set of 195 fragments was selected for the physical fragment library. The fragments were prepared as 50 mM DMSO stock solutions and transferred to 96-well polypropylene plates.
Purchased diverse fragments From the 91,379 commercially available fragments, 2000 diverse fragments were selected using the RDKit Diversity Picker node. Subsequently, k-means clustering into 500 clusters was applied to the calculated distance matrix for Daylight-like topological fingerprints using the k-Medoids node. Next, we manually inspected 500 medoids and selected a set of 283 to be included in the physical fragment library.

Chelating fragments
The Brenk filter (Supplementary Table S1) without filters for phosphor and catechol was used in the previous step to prepare the subset of chelating fragments. Based on recent reviews 17,18 , we designed SMARTS patterns (Supplementary Table S2) to find fragments capable of metal ions chelation. The RDKit Substructure Filter was used in the KNIME analytics platform. When filtering with a particular SMARTS pattern resulted in more than 100 fragment chelators, the RDKit Diversity Picker node was used to select 100 diverse fragment chelators. A total of 214 fragments were then manually selected.

Phosphate bioisostere fragments
The Brenk filter (Supplementary Table S1) without filters for Michael acceptors was used in the previous step to prepare the subset of phosphate bioisosters. SMARTS patterns for phosphate bioisosteres were designed based on a review 19 . The RDKit Substructure Filter was used in the KNIME analytics platform. When filtering with a particular SMARTS pattern resulted in more than 100 phosphate bioisostere fragments, the RDKit Diversity Picker node was used to select 100 diverse fragment chelators. A total of 159 fragments were then manually selected.

Cycloserine analogues
We applied several SMARTS patterns (Supplementary Table S4) to find fragments containing similar substructures to cycloserine. The RDKit Substructure Filter was used in the KNIME analytics platform. A total of 31 cycloserine analogues were manually selected.

Docked fragments to DdlB
The final subset contained the highest scoring fragments from docking to DdlB. The DdlB protein was prepared from the X-ray structure in complex with ADP and D-cycloserine phosphate (PDB ID: 4C5A) 11 , using Protein Preparation Wizard (Schr€ odinger Suite 2020-2, Schr€ odinger, LLC, New York, NY, 2020) 29 . Briefly, hydrogen atoms were added, residues were protonated at pH 7.0, the hydrogen bonding network was refined, waters were removed, and restrained minimisation was performed. was varied at fixed concentration of D-Ala (700 mM). The concentrations of compound 3 were 0, 25, 50, 100, 250, 500, and 1000 mM. After 20 min of incubation, 100 mM of Biomol V R green reagent was added, and absorbance was read at 650 nm after 5 min. Initial data were fitted to competitive, which v is observed reaction rate, V is the maximum rate, K M is the Michaelis constant, using SigmaPlot 12.0 software. The best fit was taken as the one having the highest R 2 , the lowest standard error and the narrowest 95% confidence interval in each parameter (see Supplementary Table S6).

Mura inhibition assay
A recombinant E. coli MurA enzyme (UDP-N-acetylglucosamine enolpyruvyl transferase) was expressed in E. coli 30 . Inhibition of the enzyme was determined using an end-point malachite green assay by detecting the orthophosphate formed during the enzymatic reaction. The final mixture (50 lL) contained: 50 mM HEPES, pH 8.0, 0.005% Triton X-114, 200 lM UDP-N-acetylglucosamine, 100 lM phosphoenolpyruvate, purified MurA (diluted in 50 mM HEPES, pH 8.0), and 5 mM of each test compound dissolved in DMSO. The final DMSO concentration was 5%. After 15 min of incubation at 37 C, the reaction was terminated by adding Biomol V R reagent (100 lL), and after 5 min at room temperature the absorbance was measured at 650 nm using a microplate reader (Synergy H4, BioTek Instruments, Inc., USA). A parallel experiment without the enzyme was performed to detect insoluble compounds under the assay conditions and subtracted from each measurement. All experiments were performed in duplicates. RAs were calculated with respect to blank experiments without tested compounds and with 5% DMSO.

Phosphate-binding assay
Phosphate binding was determined by colorimetric measurement of phosphate with malachite green in the presence or absence of the compound. To 46.9 lL of buffer (50 mM HEPES, pH 8.0), 0.625 lL of 800 lM phosphate standard, and 2.5 lL of 100 mM compound DMSO stock was added. The reaction mixture was incubated for 5 min at room temperature. Then, 100 lL of Biomol V R reagent was added and after 5 min, the absorbance was measured at 650 nm using a microplate reader (Synergy H4, BioTek Instruments, Inc., USA). Background absorbance was determined in a parallel experiment without phosphate standard and subtracted from each measurement. To determine the blank value, the compound solution was replaced with pure DMSO and the background-subtracted measurements were divided by the blank value.

HRP-PR assay
The HRP-PR assay was performed according to a previously optimised procedure 31 . Briefly, to 58 lL of buffer (50 mM HEPES, 50 mM NaCl, pH 7.5), 10 lL of 2 mM compound DMSO stock solution, and 66 lL of buffer (redox-free) or 66 lL of 3 mM DTT in buffer were added. After 15 min of incubation at room temperature, 66 lL of HRP-PR detection reagent (300 mg/mL phenol red and 180 mg/mL HRP [150-250 units/mg solid] in buffer) was added and incubated for an additional 5 min at room temperature. The final concentrations were: 100 lM compound, 100 lg/mL (282 lM) phenol red, 60 lg/mL HRP, 1 mM DTT, 5% DMSO. The reaction was then quenched by adding 10 lL of 1 M NaOH solution and the absorbance was measured at 610 nm using a microplate reader (Synergy H4, BioTek Instruments, Inc., USA). To determine the blank value, the compound solution was replaced with pure DMSO. The measured absorbance for each compound was then divided by the blank value. 3-Methyltoxoflavin was used as a control compound. All reagent solutions were freshly prepared before performing the experiments.

H 2 DCFDA assay
The H 2 DCFDA assay was performed according to a previously optimised procedure 31 . Briefly, the probe H 2 DCFDA was prepared as a 5 mM stock in DMSO and diluted 10-fold to 500 mM with 0.01 M NaOH. The probe was freshly prepared for each plate and stored in the dark at room temperature for 30 min to hydrolyse the ester. To 52.5 lL of buffer (50 mM HEPES, 50 mM NaCl, pH 7.5), 7.5 lL of 2 mM compound DMSO stock solution, 75 lL of buffer (redox-free) or 75 lL of 200 lM TCEP in buffer were added, and 15 lL of 500 mM H 2 DCFDA. The final concentrations were: 100 lM compound, 50 lM H 2 DCFDA, 100 lM TCEP, and 5% of DMSO. The microplate was covered with a lid and stored in the dark at room temperature for 30 min. Fluorescence intensity was then measured using 485 nm excitation and 535 nm emission filters (Synergy H4, BioTek Instruments, Inc., USA). To determine the blank value, the compound solution was replaced with pure DMSO. The measured fluorescence for each compound was then divided by the blank value. 3-Methyltoxoflavin was used as a control compound. All reagent solutions were freshly prepared before performing the experiments.

Resazurin assay
The resazurin assay was performed according to a previously optimised procedure 31 . Briefly, to 100 lL of buffer (50 mM HEPES, 50 mM NaCl, pH 7.5), 2 lL of 1, 0.1, or 0.01 mM compound DMSO stock solution, and 100 lL of resazurin solution (10 lM resazurin and 200 lM DTT in buffer) were added. The final concentrations were: 0.1, 1, or 10 lM compound, 5 lM resazurin, 100 lM DTT, and 1% of DMSO. The microplate was covered with a lid and stored in the dark at room temperature for 30 min. Fluorescence intensity was then measured using 560 nm excitation and 590 nm emission filters (Synergy H4, BioTek Instruments, Inc., USA). To determine the blank value, the compound solution was replaced with pure DMSO. The measured fluorescence for each compound was then divided by the blank value. 3-Methyltoxoflavin was used as a control compound. All reagent solutions were freshly prepared before performing the experiments.

Thiol reactivity assay
The assay was performed as previously described 31 . Briefly, 100 lM of compound was incubated at 37 C with a mixture of 100 lM TCEP and 25 lM DTNB (generating 50 lM TNB 2À in situ) in buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4) with 5% final DMSO concentration. To monitor TNB 2À depletion, absorbance at 412 nm was measured every 5 min for 12 h (Synergy H4, BioTek Instruments, Inc., USA). Compound background absorbance was subtracted from each measurement. 2-Chloro-N-(3-chlorophenyl)acetamide was used as a control compound. All reagent solutions were freshly prepared before performing the experiments.

Surface plasmon resonance
The SPR measurements were performed at the Infrastructural Centre for Analysis of Molecular Interactions at the Department of Biology, University of Ljubljana on a Biacore T200 (GE Healthcare) at 25 C. To assay the interaction of 3 compound with the DdlB protein we immobilised approximately 7700 RU of the purified DdlB protein on the carboxymethyl dextran-coated gold surface of the series S sensor chip CM5 (GE Healthcare) to which DdlB was immobilised via the free amino groups by an amine coupling procedure 32 . For this, DdlB protein was in 10 mM sodium acetate buffer (pH 4). Compound 3 was serially diluted in running buffer composed of 50 mM HEPES (pH 7.4), 140 mM NaCl, 0.005% P20. To assay the fragment-DdlB interaction, each fragment concentration tested (ranging from 78.125 to 5000 mM) for 40 s at a flow rate of 30 lL/min and dissociation was followed for 40 s. For the regeneration of the surface of the sensor chip, 2 mM NaOH was used. As the stock of the compound was prepared in 5% DMSO, we referenced the sensorgrams for the buffer response containing identical concentration of DMSO as the diluted sample of the compound. Furthermore, sensorgrams were referenced for the untreated surface flow-cell 1 response. The data were analysed with the Biacore T200 Evaluation Software and K D was determined by fitting the data to the steady state affinity model. The average K D s and standard deviations were determined from three titrations of 3.

Microbiological evaluation
Minimum inhibitory concentrations (MICs) for 3 were determined by broth microdilution in Cation-adjusted Mueller-Hinton broth (MH) with TES, TREK Diagnostic Systems Ltd. against S. aureus ATCC 29213, E. coli ATCC 25922, E. coli N43 and E. coli D22. All of the experiments were performed in duplicate in 96-well plate format according to CLSI guidelines and European Committee for Antimicrobial Susceptibility Testing recommendations. The bacterial suspension of a specific bacterial strain was diluted with MH broth to obtain a final inoculum of 5 Â 10 5 CFU/mL in the assay. Compound 3 dissolved in DMSO and inoculum were mixed together and incubated for 20 h at 35 C. After incubation, the minimal inhibitory concentration (MIC) values were determined by visual inspection as the lowest dilution of compounds showing no turbidity. The MICs were determined against S. aureus (ATCC 29213), E. coli (ATCC 25922), E. coli N43, and E. coli D22 bacterial strains. D-Cycloserine was used as a positive control.

High-precision docking and molecular dynamics
The DdlB protein was prepared from the X-ray structure in complex with ADP and D-Ala-D-Ala (PDB ID: 4C5C) using Protein Preparation Wizard 29 (Schr€ odinger Suite 2021-1, Schr€ odinger, LLC, New York, NY, 2021). Briefly, hydrogen atoms were added, residues were protonated at pH 7.0, the hydrogen bonding network was refined, waters were removed, and restrained minimisation was performed. Only chain A was retained. The receptor grid was generated using Receptor Grid Generation with van der Waals radii scaling by 1, partial charge cut-off 0.25 (default settings), and OPLS_2005 forcefield 33 , while the co-crystallised ADP defined the active site with the inner box of 10 Å 3 and outer box of 23.2 Å 3 . The structure of 3 was prepared with LigPrep, ionised with Epik 34 (pH 7 ± 2), and additional metal binding states were added. Docking was performed using Glide XP 21 with the default settings. The top output pose was visualised in Maestro, and then redocked using QM-Polarized Ligand Docking protocol 22 with default settings, accurate QM treatment (B3LYP/LACVP Ã ), and XP precision.
For molecular dynamics, the docking pose of 3 was merged with the crystal structure, then the system was prepared with System Builder: TIP3P 35 water molecules were added up to 10 Å from the protein surface to solvate the protein in an orthorhombic box, K þ and Clions were added to neutralise the system and produce the final 0.15 M 36 concentration, and OPLS_2005 force field 33 was used for parametrisation of the macromolecule as well as the ligand. Desmond 37 was used for molecular dynamics. The default Desmond relaxation protocol (desmond_npt_relax.msj) was used for the equilibration stage of DdlB structure: (1) 100 ps of Brownian Dynamics NVT, 10 K, small timesteps, with restraints on the solute heavy atoms, (2) 12 ps NVT, 10 K, with small timesteps and restraints on the solute heavy atoms, (3) 12 ps NPT, 10 K, and restraints on the solute heavy atoms, and (4) 24 ps unrestrained NPT; followed by the production stage: 1.2 ps interval for energy, RESPA integrator with 2 fs time step, cut-off scheme at 9.0 Å, random seed, isothermal-isobaric NPT ensemble at 300 K and 1.013 bar pressure with Nose-Hoover chain thermostat and Martyna-Tobias-Klein barostat (1 and 2 ps relaxation time, respectively, with isotropic coupling). The simulation time was 100 ns with 1000 frames per trajectory. The simulation results were analysed using the built-in Desmond tools.

Compound characterisation
Compound were purchased from several vendors (ChemBridge Corporation, ChemDiv, Enamine, Maybridge, Vitas-M Laboratory, and Wuxi LabNetwork) and used as received. The identity of compounds was confirmed with nuclear magnetic resonance, recorded on a Bruker Avance III 400 MHz spectrometer, using DMSO-d 6 as a solvent. Chemical shifts are reported in parts per million (ppm), and the central peak of the residual solvent resonance at 2.50 ppm was used as the reference. The multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets), ddd (doublet doublet of doublets), tt (triplet of triplets), dt (doublet of triplets), td (triplet of doublets), and qi (quartet of doublets), coupling constants (J) quoted in Hertz (Hz), number of equivalent nuclei (by integration). To further confirm the identity of compounds, high-resolution mass spectrometry measurements were performed on a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Compound purity was determined by HPLC analysis on Thermo Scientific Dionex UltiMate 3000 modular system (Thermo Fisher Scientific Inc.) with Waters Acquity UPLC V R HSS C18 SB column (2.1 Â 50 mm, 1.8 mm) thermostated at 40 C, injection volume, 1 mL; flow rate, 0.3 mL/min; detector k, 220 nm and 254 nm; mobile phase A: 0.1% TFA (v/v) in water; mobile phase B: MeCN. Method: 0-9 min, 5-95% B; 9-11 min, 95% B; 11-11.5 min, 95-5% B. Compounds are >95% pure by HPLC analysis, unless stated otherwise.