The effective measurements of signals have a low thermomechanical motion spectral density (9.66 ± 0.01 fmHz) and an extensive dynamic range (118.49 ± 0.42 dB) with sub‐zeptograms resolution (0.22 ± 0.01 zg) at 300 K temperature and 1 atm. Furthermore, the functional groups of the Ti3C2Tx MXene with unique adsorption properties enable a high working range ratio of ≈3100 and excellent repeatability. This Ti3C2Tx MXene device demonstrates encouraging performance advancements over other nano‐resonators and will lead the related engineering applications including high‐sensitivity mass detectors.2D materials‐based nanoelectromechanical resonant systems with high sensitivity can precisely trace quantities of ultra‐small mass molecules and therefore are broadly applied in biological analysis, chemical sensing, and physical detection. However, conventional optical and capacitive transconductance schemes struggle to measure high‐order mode resonant effectively, which is the scientific key to further achieving higher accuracy and lower noise. In the present study, the different vibrations of monolayer Ti fmHz) and an extensive dynamic range (118.49 ± 0.42 dB) with sub‐zeptograms resolution (0.22 ± 0.01 zg).A monolayer MXene nanoelectromechanical piezo‐resonator is reported for the first time, achieving high‐resolution molecular sensing performance in high order mode. The effective measurements of signals have a low thermomechanical motion spectral density (9.66 ± 0.01  However, the shortages of the sensitive vibration transduction and the signal acquisition technology at the atomic level remain urgent obstacles in the practical development processes. It is widely believed that higher resonant frequencies, as well as the quality factor (Q‐factor), can effectively facilitate the development of resonators with enhanced signals at the atomic scale. Several monolayer materials, such as transition metal dichalcogenides (TMDCs) and graphene, are expected to achieve high operating performance due to their near‐limit surface‐to‐volume ratios and robust mechanical properties and have sparked several high‐performance resonators. However, the relatively low surface adsorption capacity and high noise hinder the nano‐resonators as ultra‐high precision detectors with complex functionalization processes. For the Ti3C2Tx MXene with the high electrical performance and mechanical properties in monolayer states, the extensive presence of tunable functional groups on the Ti3C2Tx MXene provides unique features that are different from previous 2D materials, allowing more efficient adsorption of molecules as well as more sensitive ultra‐weak signals detection. The non‐uniformly distributed mechanical and piezoelectricity of the Ti3C2Tx MXene can potentially realize resonance signal detection of higher‐order vibrational modes at the atomic level in a resonant system. Some of the works predict the special behavior of the MXene as a high‐performance and high‐sensitivity resonator for applications. However, the exploration and implementation of the Ti3C2Tx MXene‐based nanomechanical resonance systems have not been elaborated to date yet.The imperative demand for stable, sensitive, and efficient nanomechanical resonant systems has triggered research into materials, structures, and methods to perform studies for high‐sensitivity sensors and condensed matter physics.2, 33C2Tx MXene are investigated by constructing a high‐sensitivity piezoelectric transduction scheme based on the piezoelectric response of the asymmetric atomic structure. The high‐precision piezoelectrical method provides a more accurate resonance detection than that of conventional optical and capacitive detections. Devices driven by alternating current (AC) signals show a standard resonant waveform for the characteristic f2,3 resonant mode. The high‐performance molecular adsorption properties and the resonance responses of the Ti3C2Tx MXene are systematically investigated, and demonstrate ultra‐high sensitivity and reproducible multi‐molecular detection with a large dynamic range (DR). These studies report the resonance properties of the Ti3C2Tx MXene for the first time and reveal the low noise signal and high mass resolution due to the unique structure of the Ti3C2Tx MXene.Here, the resonant properties of the Ti23C2Tx MXene mechanical resonator, in Figure 3C2Tx MXene flake on a circular‐hole vibrating cavity, source and drain electrodes, an insulating layer, and a local gate, respectively. The high‐quality synthetic Ti3C2Tx MXene can be effectively transferred to a pre‐patterned hole SiO2/Si substrate  voltage is applied to the conductive Si layer as the local gate voltage to generate an electrostatic force and provides the strain to the Ti3C2Tx MXene in elastic elongation and electrostatic bias states. This AC drive power supply can also apply a frequency‐modulated voltage to detect high‐frequency mechanical motion at a lower mixed frequency, avoiding difficulties in direct radio frequency response from impedance mismatches and parasitic effects. The use of a lock‐in amplifier allows the mixed‐down current generated by the Ti3C2Tx MXene resonator as a demodulator to test the resonator amplitude at the nanometer scale with radio frequencies. Figure 3C2Tx MXene. The unique multi‐atomic layer consists of the central layer and outer layer of titanium (Ti) atoms as well as the upper and lower carbon (C) atom layers encapsulated by the Ti atom layers. The outer layer of Ti atoms provides a large number of functional group sites to be attached. To fabricate the high‐quality Ti3C2Tx MXene, the minimally intensive layer delamination (MILD) method is used to prepare monolayer MXene flakes with a large‐scale flat area , and X‐ray photoelectron spectroscopy (XPS) are used to characterize the quality of the synthetic monolayer Ti3C2Tx MXene, indicating the corresponding elemental composition, etching purity, and functional group composition. The Raman spectrum of the Ti3C2Tx MXene is shown in Figure −1. The vibration at 204 cm−1 corresponds to the vibration A1g of Ti, C, and surface functional groups, while the vibration at 734 cm−1 produces a redshift concerning the solution or film as the vibration A1g of C atoms. As the XRD is shown in Figure 3C2Tx MXene has a little (104) peak at ≈39°and the (002) peak of Ti3C2Tx MXene moves forward to ≈6.5°compared with Ti3AlC2 MAX phase, corresponding to a C lattice parameter of 27.1 Å, confirming the high purity of the sample. In Figure 3C2Tx are analyzed by an XPS. According to the XPS spectrum, the dominated peaks of Ti and C indicate the high purity of the Ti3C2Tx MXene in the fluoride etching strategy. And in Figure  In Figure 3C2Tx MXene samples, with the surface functional group ‐OH.The basic structure of the Tin Figure 10 A d3C2Tx MXene. This modulation scheme is mainly from the piezoelectric response current based on strain‐induced polarization charges, and the mechanical vibration of the Ti3C2Tx MXene is variable to control the output piezoelectric electric signal. For the Au‐MXene‐Au structure, the Fermi energy level is flat with an unstretched state in the upper of Figure 3C2Tx MXene is stretched in a resonant state, while the piezoelectric polarization charge accumulates at the source and drain ends. These charges can raise and reduce the height of the potential barriers as the lower in Figure  In the AC signal‐driven Ti3C2Tx MXene resonator, the resonant amplitude maintains a regular motion in response to the AC frequency and the gate DC bias. This continuous signal drives the Ti3C2Tx MXene to generate a varying piezoelectric potential, which affects the potential distribution in the resonator . The current‐voltage (I–V) curve of the monolayer Ti3C2Tx MXene device produces asymmetric transmission characteristics, which are modulated by piezoelectric polarized charges from the tensile strain. This phenomenon is responded to the piezoelectricity as the reported works with tungstite and zincite structures, whereby piezoelectric polarization is the main cause of modulated changes in carrier transport properties. Furthermore, the configuration of the metal‐piezoelectric materials and the intrinsic asymmetric modulation process can introduce a transduction scheme for the strain‐induced polarization phenomenon to sense external signals. In Figure 3C2Tx MXene can be always in a cyclic strain process as stretch‐recovery‐stretch from the crest to the trough during the resonance. This process leads to the formation of the piezoelectric polarization and eventually causes the accumulated charges at the source and drain. Figure 3C2Tx MXene resonator, where the responses of the piezoelectric current (short circuit) and the voltage (open circuit) are measured by a DC gate driving bias. The generation of polarized charges varies with the degree of the deformation, resulting in a piezoelectric output with the deformation cycles. When the Ti3C2Tx MXene layer is removed from its initial position and begins to stretch, the piezoelectric polarization charges accumulate and flow to the external circuit. When the layer is stretched to its limit and begins to shrink, the reversed piezoelectric polarization current flows back to form the negative response current, which directly demonstrates the conversion of the mechanical deformation to the output currents or voltages. Moreover, the experiments are also conducted on the piezoelectric output produced by different numbers of layers as shown in Figure As the MXene resonator, the metal‐MXene‐metal structure allows resonant deformation to produce piezo‐electric transport characteristics through the vibrations of the TiTx MXene.8 This3C2Tx MXene layer are tested to analyze the characteristics of the system in a sealed nitrogen (N2) environment at room temperature (300 K). The Ti3C2Tx MXene layer is driven by an AC drive signal VwcFM with a 200‐mV source bias, causing the resonator to vibrate with a time‐varying electrostatic force at the frequency ωc. The Ti3C2Tx MXene resonator acts as a demodulator to trans conduct the information stored in the amplitude and generates a time‐varying piezoelectric output as a mixed current mixI, detected by the lock‐in amplifier for enabling nanoscale amplitude detection at radio frequencies. By scanning the driving frequency, the frequency response curve of the monolayer Ti3C2Tx MXene resonator is obtained and the multi‐response modes can be identified as shown in Figure 3C2Tx MXene resonator can generate measurable piezoelectrical signals in the range of f0,2 = 90.64 MHz, f1,3 = 206.89 MHz, and f2,3 = 234.59 MHz as well as other relatively weak modes. That is because only while the piezo‐potentials are strong enough (the strain > 1.05% in the Ti3C2Tx MXene) to overcome the neutralized free electrons due to the high conductivity of the Ti3C2Tx MXene, the piezo‐signals can be effectively detected by the peripheral circuit system. The insets of Figure 0,2, f1,3, and f2,3.  in 1 atm. In Figure 2,3 mode exhibits an out‐of‐scale strong signal, the measured output piezo‐current has a 121 ± 4% enhancement while the corresponding strain is only 70% improvement (related to 54% piezo‐current increase) from f1,3 to f2,3 mode. This curious phenomenon may be caused by the special piezo‐structure of the Ti3C2Tx MXene, which leads to a non‐uniform distribution of the piezoelectric signal between the atomic layers  × 1010s−1. Figure 3C2Tx MXene resonator with the 200 mV AC drive bias. The amplitude can be adjusted by changing the gate voltage, and the characteristic patterns with different gate voltages are obtained. The effect of the gate voltage on the Ti3C2Tx MXene resonator can be described as∂Cg∂x is the spatial derivative of the gate capacitance, gV is the static voltage applied to the local gate electrode, and sdV is the AC voltage between the source and drain electrodes. A larger gate voltage will result in a more intense amplitude deformation and cause a stronger piezoelectric response in the Ti3C2Tx MXene, and the detected resonant response current is able to be more pronounced. (Part Vg < 1.5 V), the resonant frequency exhibits a symmetrical peak signal, which is directly related to the pretension (T) of the circular membrane. The pretension in the f2,3 vibration mode is extracted asρ is the mass density, b is the ratio of higher‐order modal frequency to fundamental frequency, r is the effective radius of the resonator, and h is the thickness of the Ti3C2Tx MXene layer. The pretension of the Ti3C2Tx MXene layer is estimated to be ≈0.50 ± 0.01 N m−1. Nanomechanical resonators with high‐aspect ratios will enter a nonlinear regime during high amplitude motion. As Vg increases to 2 V, the resonance peak changes to an asymmetric one from a symmetric line shape, confirming the Ti3C2Tx MXene resonator has become the nonlinear regime. In this case, it can be described using the Duffing resonator model with an effective additional term αmx3 and ηmx2x˙, which is related to the nonlinear restoring force and nonlinear damping, and the detailed derivation is shown in the Part x is the motional amplitude, k is the effective spring constant, m is the resonator mass, γ is the linear damping coefficient, η is the nonlinear damping coefficient, α is the Duffing force coefficient, F0 is the amplitude of the driving force, ω is the driving frequency, and t is time. The duffing force originates from the nonlinearities and the external electrostatic. When Vg increases from 1.5 to 2 V, the increase in electrostatic forces acting on the Ti3C2Tx MXene resonator leads to a greater amplitude of vibration, and the Duffing force dominates the vibration process, leading to distortion of the peak shape and bi‐stability. We can estimate the effective damping γ from the analytical solution of the linear resonant resonator equation of motion aseffM is the theoretical effective mass of circular resonator expressed as Meff=18πd2hρ and f2,3 is the resonant frequency. The effective damping γ of the linear Ti3C2Tx MXene resonant resonator is estimated as (9.94±0.01)×10−10kgs. For a circular Ti3C2Tx MXene membrane, the duffing‐type nonlinear coefficient k can be expressed asv is the Poisson's ratio of the Ti3C2Tx MXene, and ε is the initial strain. The critical amplitude ca determines the onset of bistability and is given byTo study the basic performance of the Ti2,3 mode is estimated as 4.14 ± 0.12 nm. The thermomechanical motion spectral density at the resonant frequency f2,3 is given bymw is the angular resonant frequency. Based on the structure of the circular hole resonance device, the fundamental mode of thermomechanical motion out of the plane is most prominent. The thermomechanical motion is the lowest level of motion mode possible to be measured by the device and expresses the detection limit. The thermomechanical motion spectral density is defined as Sx,th12=9.66±0.01 fmHz, and achieved superior performance relative to graphene, molybdenum disulfide (MoS2), and black phosphorus (BP). The dynamic range (DR), which is defined as the ratio between the highest signal level prior to any nonlinear bifurcation and the lowest detectable level, can be described asf is the measurement bandwidth. The DR of the monolayer Ti3C2Tx MXene resonator is 118.49 ± 0.42 dB, which is higher than the nanomechanical resonators based on the graphene, MoS2, and boron nitride (BN). The increased gate voltage leads to a DC electrostatic force between the Ti3C2Tx MXene layer and the local gate, and the generation of the additional tension lifts the resonant frequency. Therefore, the gate voltage is used to adjust the resonant frequency in atomically thin resonators. The effect of the gate voltage on the resonant frequency of the monolayer Ti3C2Tx MXene resonator is indicated in Figure  More simulations of this phenomenon , the measured Young's modulus tends to be stable at 0.38 TPa with the increase of thickness, while the measured values are dramatically increasing as N is decreasing from 3 to 1. Since high‐quality single‐crystal Ti3C2Tx MXenes with few defects are used in the experiments, it is believed that the special size dependence of Ti3C2Tx MXene with a large surface volume ratio is due to the surface modification of the material, which makes the surface effect obvious. Extensive theoretical and experimental results show that the free surface of nanomaterials can undergo a significant relaxation phenomenon characterized by bond length contraction, and the bond shrinkage can significantly affect several atomic layers below the surface of the material. The elastic constants of crystals depend on the distance between the interatomic, so the surface elasticity of crystals is greatly affected by surface relaxation. A Ti3C2Tx MXene containing modified surface layers can be regarded as the composite flake shown in the inset of Figure 0 and a surface layer with modulus Es which is correlated to the surface bond length contractions. It is worth noting that the influence brought by the surface effect extends from the surface to the interior, gradually decreasing and eventually subsiding; however, a reasonable simplification can help in the analysis and understanding of the problem, in which the relaxed surface layer is approximately disposed of as a uniform shell. Consequently, a sandwich composite layer model is used to simplify the analysis of size‐dependent elastic properties. From the experimental results and simplified models, the repeating element with ts = 0.49 nm obtained through curve fitting implies that a Ti3C2Tx MXene with one layer (0.98 nm) will be fully relaxed and have an effective modulus equal to 5.90 ± 0.28 TPa, and this model can effectively describe the elastic state from layer 1 to bulk.  far exceeds that of graphene (−1.96 × 10−9 N with acetone), MoS2 (−1.26 × 10−9 N with acetone), and BP (−2.12 × 10−10 N with acetone), providing great potential for small molecule signal detection applications ,57 MoS3C2Tx MXene resonator. The effects of propanal, ammonia, ethanol, and acetone on the resonant frequencies are also tested at 100 ppm concentrations as shown in Figure 3 acetone molecules, ≈ (6.63 ± 0.11) × 103 propanal molecules, ≈(7.54 ± 0.08) × 103 ethanol molecules, and ≈(1.42 ± 0.14) × 104 ammonia molecules. It is believed that this quantitative difference stems from target molecules adsorbing to the Ti3C2Tx MXene surface in different ways, with a single molecule occupying about three attachment sites for acetone, propanal, and ethanol, and about one attachment site for a single ammonia molecule  are able to be adsorbed on one 3 µm diameter Ti2,3 resonant mode, the high response signal achieves high‐quality extraction of target information, and a high signal‐to‐noise ratio of > 35 is achieved in all kinds of detection processes. As shown in Figure 3C2Tx MXene nanomechanical resonator has the performance advantage of ultra‐high signal‐to‐noise ratio compared to conventional 2D material‐based gas sensors such as BP, MoS2, and graphene oxide (RGO). Further, we compare the thermomechanical motion spectral density of various nanomechanical resonators to measure the performance of the Ti3C2Tx MXene nanoelectromechanical resonators in Figure  For our Ti3C2Tx MXene resonators, the mass resolution δm of the resonator can be derived asAs a small molecular detector, the signal‐to‐noise ratio is an important index to evaluate its performance, and the goal of the low interference signal is achieved by using the self‐excellent filtering effect on noise signals from the high conductivity and mixing detection techniques (The details are provided in Part de (RGO).61 Furδm = 0.22 ± 0.01zg ([0.22 ± 0.01] ×10−21g), and the working range ratio of the saturated detectable mass to minimum mass resolution is up to ≈3100. This mass resolution is a prominent performance improvement compared to previously reported nanomechanical resonators as shown in Figure 3C2Tx MXene relative to other materials (See Part  Table 3C2Tx MXene resonator and that of other reported nano‐resonators (including any pressure and temperature conditions). The monolayer Ti3C2Tx MXene not only exhibits the smallest mass resolution but also shows an excellent thermomechanical motion spectral density, which bodes well for the potential applications in microscopic signal detections.Here, the ultra‐high mass resolution can be obtained as 33C2Tx MXene nanoelectromechanical resonator are realized by the piezoelectric effect and the mixing detection technique. The atomically thin resonator exhibits a strong characteristic f2,3 mode response signal in the high‐frequency range, which is attributed to the special piezoelectric distribution of the 2D Ti3C2Tx MXene layer with low noise and large dynamic range. In addition, the functional groups of the Ti3C2Tx MXene play important roles in the sensing detection of target molecules with high sensitivity and large working range, revealing the working mechanism and contribution of atomic structures in molecular adsorption and low mass resolution sensing performance. This study can demonstrate the promise of the Ti3C2Tx MXene for chemical, biological and scanned‐probe sensing.In conclusion, the high order resonance characteristics of the monolayer Ti43C2Tx MXene resonator consists of two parts: monolayer MXene transfer and electrode preparation. PDMS is used to transfer the monolayer MXene to the prefabricated cavity, and the morphology is confirmed by optical microscopy and atomic force microscopy (AFM). Photoresist  is rotated on Si wafers with SiO2 oxide layer and pre‐baked at 115 ℃ for 1 min. The mask is aligned with the monolayer Ti3C2Tx MXene for electrode exposure, and the samples are rinsed with the developer and deionized water for 60 and 60 s, respectively. The samples are calcined at 90 °C for 1 min. Standard electrodes (5 nm Cr and 50 nm Au) are prepared by physical vapor deposition (PVD) to remove excess photoresist and metal layers. Connect external wires and seal the unit with a nitrogen‐filled chamber. See Parts The preparation of the monolayer Ti3C2Tx MXene in elastic elongation and electrostatic bias states. This AC drive power supply can also apply a frequency‐modulated voltage to detect high‐frequency mechanical motion at a lower mixed frequency, avoiding difficulties in direct radio frequency response from impedance mismatches and parasitic effects. The use of a lock‐in amplifier allows the mixed‐down current generated by the Ti3C2Tx MXene resonator as a demodulator to test the resonator amplitude at the nanometer scale with radio frequencies.A designed mixing piezo‐detection technique is used to measure the vibrational frequency response. The output signal is transferred to a lower frequency without losing the stored information in its amplitude, thus achieving more convenient detection. A DC voltage is applied to the conductive Si layer as the local gate voltage to generate an electrostatic force and provides the strain to the Tip < 0.05 is considered statistically significant. To further investigate the signal noise, the Allan deviation is used to describe the current noise at half of the maximum amplitude (Part n) for each statistical analysis is detailed in the legend. Statistical analysis is performed using MATLAB.The following steps are conducted for data preprocessing. Before starting the experimental steps, the sample quality is checked to obtain valid raw data. Origin software is used for statistical analysis and graphical representations of data. Data are expressed as mean ± standard error and come from three independent sets of experimental results. One‐way and two‐way analysis of variance (ANOVA) Tukey's test is used to examine the frequency distribution of different layers of MXene resonators. A The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Reaction of the new electron‐rich diphosphine 1,3‐bis((di‐tert‐butylphosphaneyl)methyl)‐2,3‐dihydro‐1H‐benzo[d]imidazole (1) with [PdCl2(cod)] occurred in a stepwise fashion, first by single C−H bond activation yielding the alkyl pincer complex [PdCl(PCsp3HP)] (3) with two trans phosphane donors and a covalent Pd−Csp3bond. Activation of the C−H bond of the resulting α‐methine Csp3H−M group occurred subsequently when 3 was treated with HCl to yield the NHC pincer complex [PdCl(PCNHCP)]Cl (2). Treatment of 1 with [NiBr2(dme)] also afforded a NHC pincer complex, [NiBr(PCNHCP)]Br (6), but the reactions leading to the double geminal C−H bond activation of the N−CH2−N group were too fast to allow identification or isolation of an intermediate analogous to 3. The determination of six crystal structures, the isolation of reaction intermediates and DFT calculations provided the basis for suggesting the mechanism of the stepwise transformation of a N−CH2−N moiety in the N−CNHC−N unit of NHC pincer complexes and explain the key differences observed between the Pd and Ni chemistries.We report the first examples of metal‐promoted double geminal activation of C(sp Agemof a reaction: The first examples of stepwise (Pd) or direct (Ni) metal‐promoted double geminal activation of Csp3−H bonds leading to imidazole‐type N‐heterocyclic carbene metal pincer complexes under mild conditions are presented. Pincer‐type ligands, defined as tridentate ligands that bind to a metal centre in a meridional fashion, have become ubiquitous in various branches of chemistry, largely because of the easy tunability of their stereoelectronic properties.4, 5 Tsp2(2)−H bond across a zerovalent metal precursor, which was introduced about 20 years ago.imidazole  carbene derivatives have been obtained recently,[H‐benzo[d]imidazole, its corresponding azolium salt and some palladium and rhodium PCNHCP pincer complexes.We first set out to prepare an electron‐rich diphosphine as potential precursor to a PCrecently,90, 911 was treated with [PdCl2(cod)] in CH2Cl2 at room temperature overnight, a white solid 2 was obtained in 70 % yield. Its 1H NMR spectrum contained no signal corresponding to the NCH2N protons of 1 and 31P NMR spectroscopy confirmed P‐coordination to Pd. An X‐ray diffraction analysis established the formulation of 2 as a NHC pincer complex, [PdCl(PCNHCP)]Cl, resulting from the double geminal C−H bond activation of the N−CH2−N unit of 1  (3) was confirmed by X‐ray diffraction analysis 4] and an imidazolium chloride precursor containing two lateral diphenylphosphino‐substituted o‐phenylene linkers.sp3−Pd bond distance of 2.0747(12) Å in this complex is very similar to that observed in 3. In both cases, the chloride ligand contributes to the stabilization of the complex. It is noteworthy that the preference for alkyl versus NHC coordination has been rarely encountered in structurally characterized examples  in Scheme 2−N hydrogen atom in 1 is formally eliminated as H+ whereas the remaining methine N−CH−N hydrogen in 3 behaves as a hydride, reacting with acids. In this context, it is interesting that with their PCalkylP PdII pincer system C 2 and PPh3 in THF, dehydrohalogenation of A afforded the pincer complex B with a Ni=Csp2double bond. (4) occurred crown‐5, to afford [PdN3(PCsp3HP)] (5) [Eq. (5)], which was characterized by X‐ray diffraction  and displays a bent azide ligand (Pd1−N3−N4 120.3(2)°). Photochemical irradiation  of this complex did not result in liberation of N2. When a benzene solution of 3 was exposed to air, the solution turned purple within seconds, but the corresponding highly soluble compound could not be characterized. The only peak in its mass spectrum that could be assigned corresponded to 3‐H. This reaction was accompanied by the formation of 2, probably as a result of partial decomposition and liberation of HCl, that would readily protonate unreacted 3. Similarly, a purple colour developed when a benzene solution of 5 was exposed to air. In contrast, a solution of the pincer complex 4 in benzene or pentane was air‐stable for at least 0.5 h, although the methyl ligand renders this complex more electron‐rich. In attempts to better characterize the purple compound resulting from exposure of 3 to air, we prepared the analogue of 3 from the diphenylphosphino‐substituted ligand 1Ph2,1, Csp3−H bond activation occurred upon reaction with [PdCl2(cod)] to give 3Ph2. However, this complex did not change colour upon exposure of its solution to air, which highlights the critical role of the strong electron‐donating tBu substituents in 3 which make the metal more electron‐rich and thus much more air‐sensitive.The crystal structure of 4 Figure  is simil2 double activation by DFT methods. Starting from the adduct I1bis, the successive steps are shown in Figure 1 on [PdCl2(cod)] to give first an intermediate I1 in which both the cod ligand and 1 are monodentate  and results in several conformers from which different reaction paths are conceivable. Three mechanisms were tested for the CH2 double activation reaction. The first one starts from I1bis  in which the halides are in cis position, the fourth site of the palladium square planar coordination sphere being occupied by an agostic interaction (Pd−H distance of 1.732 Å) involving one hydrogen atom of the CH2 group, which strongly weakens the C−H bond (1.246 Å). This step opens the way to the hydride transfer yielding an imidazolium zwitterionic complex I2 through a low transition state TS1 . The intermediate I3 evolves to give I3bis in which the free chloride anion interacts with the Pd‐bound hydride. This interaction becomes stronger and TS3 is associated with a barrier of 9.4 kcal ⋅ mol−1 (TS3‐I3bis) and results in the removal of this ligand as a proton, in a formal redox reaction. Thus, the electronic doublet ensuring the Pd−H bond is transferred to the imidazolium moiety that is reduced to an imidazolate. This step is mediated by the palladium and a Pd−C bond is formed with the imidazolate. In the final step, the HCl fully liberated (I4 to I4bis) attacks the N−CH−N hydrogen to give the final product I5 with liberation of H2. Consistent with the key role of HCl in this step, its trapping by a base stops the reaction at the intermediate stage I4. This is consistent with the relatively high barrier (TS4‐I4) of 24.8 kcal ⋅ mol−1 associated to TS4 compared to the previous steps: the amine traps the acid before it reacts. It should be noticed that not only the barrier associated to TS4 is high but the reaction is also endergonic, I5  being less stable than I4 . The overall exergonicity of the reaction is ensured by the H2 departure that stabilizes the system by −4.6 kcal ⋅ mol−1 .This step is exergonic  (path thereafter denoted as OX) which involves metal insertion into a C−H bond of the CH2 group to give formally a PdIV species. We also explored the feasibility of a cyclometallation–deprotonation mechanism (path hereafter denoted CMD), in which a C−H atom is eliminated as HCl with the chloride ligand.I2CMD is equivalent to I4bis and I2ox will undergo a reductive elimination also to give an equivalent of I4bis. The final step of these two alternative mechanisms will be the same as that of Figure I4bis. Most importantly, the energy associated with TS1 is higher for the OX and CMD mechanisms than for that of Figure OXTS1 and CMDTS1) are even higher in energy than all those computed for the first proposed path, making them non‐competitive. Consequently, we can exclude these mechanisms (OX and CMD) for the double CH2 bond activation with palladium under our reaction conditions. Note that reactions following a typical CMD mechanism usually involve a carboxylate ligand which remains coordinated to the metal at the end of the reaction, in contrast to the chloride ligand that dissociates from the metal centre.We then investigated two alternative mechanisms for CH1 with [NiBr2(dme)] in THF at room temperature and observed a very fast reaction leading directly to the NHC pincer complex [NiBr(PCNHCP)]Br (6) ], we examined the reaction of ) Scheme . It was 2 Figure .II, the reactions leading to the double Csp3−H bond activation of the N−CH2−N moiety of 1 were too rapid to allow the observation or isolation of an intermediate with a covalent Csp3−Ni bond, even in the presence of NEt3 (in CD2Cl2 or C6D6). In contrast, the PdII intermediate 4 was readily isolated , but in contrast to the behaviour of the PdCsp3−H bond in 3.With Nid Scheme , which dII centre by the organic ligand is slightly stronger  than that of PdII  is slightly higher with Pd than with Ni . The trend is reversed for the next two steps, TS2 and TS3, since the barriers are higher for Ni  than for Pd , respectively. However, the energy barrier for the last step (I4 to TS4) is significantly higher for Pd  than for Ni . This step is rate determining in the case of palladium but not of nickel, for which it is associated to TS3. These values explain the experimental findings. The highest barrier for palladium is larger than that for nickel, consequently the reaction will be faster for the latter. The small energy barrier associated to TS4 for nickel explains the failure to trap the hydride intermediate: the reaction is too fast, and the addition of an amine cannot prevent the acid of reacting. On the contrary, the high barrier in the case of palladium allows trapping of the intermediate. Another difference is the endergonicity of the reaction with palladium, I5 is less stable than I4bis, the exergonicity of the reaction being ensured by the departure of the H2 molecule. This is not the case for nickel where I5 is more stable than I4bis.For comparison, we also computed the reaction mechanism with Ni and the overall mechanism was found to be similar to that with Pd. However, significant differences were observed in the energies involved. The first steps are very similar in energy, the complexation of the Ni1 as precursors to PCNHCP pincer systems, coordination of the P donors to the MX2 unit is most likely to occur first, and this brings the CH2 group to be activated in closer proximity to the metal M. This step could be further assisted by an additional interaction between a N atom of the heterocycle and the metal centre,sp3−H bond to be activated, resulting in a Csp3−M bond. This step would be followed by reductive‐elimination of HX, or σ‐bond metathesis between a C−H and a M−X bond, or by dissociation of a halide followed by anion‐promoted deprotonation. These possibilities have been examined recently.With diphosphines such as sp3−H bonds, this work illustrates major differences between pincer ligands bearing a NHC donor in the bridgehead position but different side arms (phosphines vs. imines) and for a given pincer ligand, the differences between the metal centres, Ni being generally far more reactive than Pd, although the latter sometimes allows, like in this study, the isolation of intermediates that shed light on the course of important and catalytically relevant reactions, such as Csp3−H bond activation and NHC formation.In addition to providing new insights into the double geminal activation of Csp3−H activation can lead to metal carbene complexes,[2−N moiety of an imidazole‐type heterocycle, directly affording a coordinated NHC ligand under mild conditions. Such facile, room‐temperature metal‐promoted access to highly popular NHC‐metal complexes offers considerable synthetic potential as readily accessible and stable NiII or PdII metal reagents were used. Thanks to their versatile chemistry and flexible coordination sphere, both NiII or PdII complexes were competent to perform the double Csp3−H bond activation, but the reactions were too fast with NiII to identify any intermediate. With PdII, in contrast, a key alkyl intermediate resulting from single Csp3−H bond activation was isolated and fully characterized. As discussed in this paper, several examples have shown that the first step of the Csp3−H bond activation of a N−CH2−N moiety involves the formation of a metal alkyl complex. The pathways leading to the activation of the resulting methine Csp3H−M group have been generally less discussed, probably because such intermediates have more rarely been isolated. Our combined theoretical and experimental study has provided new insights into the mechanism of the double geminal activation of the Csp3−H bonds of a N−CH2−N moiety and shown that the properties and reactivity of the Csp3H−M bond generated in the first step of the reaction depend very much on subtle effects. Overall, the double Csp3−H activation of the N−CH2−N hydrogen atoms of 1 can be formally viewed as the metal‐ and chelate‐assisted successive elimination of H+ and H−. Our results extend to PCNHCP chemistry the double Csp3−H activation approach leading to PCsp2P pincer complexes for which the first C−H activation step occurs by oxidative addition or electrophilic activation, and the subsequent activation of the α‐methine group by α‐hydride elimination, hydride or proton abstraction.Although it was known that metal‐promoted double C2−N moiety, without the need to prepare an azolium derivative or a free carbene, and of using air stable and readily available metal precursors, will facilitate the study of their reactivity.Future work should explore the scope of the protocols described in this work to access diverse NHC pincer complexes. The associated advantages of starting directly from one of the many neutral heterocyclic candidates containing a N−CH1), 2115915 (2⋅CH2Cl2), 2115916 (3⋅2 C6H6), 2115917 (4), 2115918 (5) and 2115919 (6)2115914  should be addressed to the authors.Supporting InformationClick here for additional data file.


II6L4 coordination cage can efficiently encapsulate both resorufin and resazurin in the form of dimers, dramatically modulating their optical properties. Furthermore, binding within the cage significantly decreases the reduction rate of resazurin to resorufin, and the rate of the subsequent reduction of resorufin to dihydroresorufin. During our studies, we also found that upon dilution, the PdII6L4 cage disassembles to afford PdII2L2 species, which lacks the ability to form inclusion complexes – a process that can be reversed upon the addition of the strongly binding resorufin/resazurin guests. We expect that the herein disclosed ability of a water-soluble cage to reversibly modulate the optical and chemical properties of a molecular redox probe will expand the versatility of synthetic fluorescent probes in biologically relevant environments.Confining molecules within well-defined nanosized spaces can profoundly alter their physicochemical characteristics. For example, the controlled aggregation of chromophores into discrete oligomers has been shown to tune their optical properties whereas encapsulation of reactive species within molecular hosts can increase their stability. The resazurin/resorufin pair has been widely used for detecting redox processes in biological settings; yet, how tight confinement affects the properties of these two dyes remains to be explored. Here, we show that a flexible Pd Water-soluble nanocontainers that can encapsulate and modulate the properties of synthetic fluorescent probes could expand the versatility of such probes in biologically relevant environments. Here, the authors encapsulate the resazurin/resorufin redox pair within a flexible palladium-based coordination cage and find that confinement drastically alters the optical and redox properties of the two dyes. As early as 1945, Twigg proposed the use of resazurin (nowadays widely known as Alamar Blue) for monitoring bacterial and yeast contamination of milk4. On a larger scale, resazurin has been used to investigate metabolic activity within microbial communities in large aquatic ecosystems6 and to monitor biological activity in sewage treatment plants7. Resazurin has also been used to detect enzymes  whose activity results in the production of reducing agents, which can then induce the formation of the fluorescent resorufin9. Owing to the high emission intensity of resorufin, the resazurin/resorufin pair has been employed for detecting and studying corrosion10 and other important processes14 on very small scales using fluorescence microscopy. Additionally, fluorescence microscopy can provide important insights into the properties of supported catalysts capable of facilitating the resazurin→resorufin reduction19. However, despite several decades of research on resazurin and resorufin, little attention has been devoted to the behavior of these two dyes within confined environments.The resazurin/resorufin pair has long been studied and utilized as a structurally simple system for detecting reducing environments. Upon reduction, the weakly fluorescent dye resazurin is converted into strongly emissive resorufin. This reaction is readily induced by metabolically active cells and can thus serve as a method for evaluating cell viability25. For example, the oxidation potential of tetrathiafulvalene could be shifted considerably upon encircling it within macrocyclic “hosts”, such as α-cyclodextrin24 or cyclobis(paraquat-p-phenylene)25. Similarly, the optical properties of dyes can be modulated by confining them within the cavities of self-assembled cages29. Surprisingly, however, little attention has been devoted to the behavior of resorufin and resazurin in confined spaces, and no strong confinement effects for these two dyes have been reported to date. For example, the fluorescence of resorufin was not markedly affected by encapsulation within β-cyclodextrin30 or within a tetralactam macrocycle incorporating two anthracene moieties31. Similarly, resorufin placed within micelles32, microemulsion droplets33, and one-dimensional channels of metal–organic frameworks34 retained its strong emission.Placing optically active and/or redox-active species under confinement can profoundly affect their physicochemical characteristics35 ) and four triimidazolylbenzene (TImB) ligands. Uniquely, 1 combines a well-defined cavity size with sufficient37 structural flexibility to simultaneously accommodate38 two small aromatic guest molecules as diverse as azobenzenes,40 BODIPY dyes41 and pyrene35. The encapsulation of these molecules is driven by the hydrophobic effect and their attractive interactions with the cage through a combination of π–π stacking and van der Waals interactions, with additional contributions from other, weaker interactions such as dispersion forces. The binding is further stabilized by attractive interactions between the two guest molecules, which reside near each other.Here, we investigated the host–guest complexation of resorufin and resazurin within a cationic cage based on Pd–imidazole coordination1. Moreover, in contrast to the above guests , both resorufin and resazurin are monoanionic, strengthening binding through Coulombic interactions with the duodecuply  charged host. Indeed, we found that cage 1 can bind two molecules of both dyes, thus inducing noncovalent dimerization, which dramatically changes their optical properties, causing strong shifts in the absorption spectra and effectively turning off the emission of resorufin. Furthermore, encapsulation within the cage markedly decreases the kinetics of the reduction of resazurin to resorufin, and the further reduction of resorufin to dihydroresorufin. Our results indicate that confinement effects, ubiquitous in nature, have a profound effect on the behavior of a widely used redox probe.We hypothesized that, owing to their structural resemblance to the previously studied guests, resorufin and resazurin could similarly be encapsulated within 2 by cage 1. The 1H nuclear magnetic resonance (NMR) spectrum of a 2:1 mixture of 2 and 1 recorded at room temperature featured relatively broad signals, hampering the characterization of the complex. We found, however, that gentle heating preserved the integrity of the inclusion complex while significantly sharpening the signals. Figure 1H NMR spectrum of the complex recorded at 320 K. The DOSY spectrum ⋅2, giving rise to an overall guest/host ratio of 3:1 ⋅2]n. This result emphasizes the unique ability of one molecule of 22⊂1 to “extract” free guest 2 from another and to incorporate it within the crystalline lattice.First, we obtained single crystals of the complex by slow water evaporation from an aqueous solution of 22⊂1, we found that each 22⊂1 moiety was surrounded by not one but four molecules of 2, which were not shared with the neighboring cages ⋅24]n  and the process of crystal formation coupled with guest extraction can be written asRemarkably, when crystallization was induced by diffusing acetone vapor into the aqueous solution of ges Fig. . Therefo2 fit well within the shallow cavities formed by the outer surfaces of the TImB panels and the peripheral TMEDA groups ⋅24, see Supplementary Fig. 22⊂1)⋅2]n or [(22⊂1)⋅24]n  was followed by the formation of colorless crystals of the empty 1. The strong preference for crystallizing [(22⊂1)⋅2]n and [(22⊂1)⋅24]n is evidenced by the formation of these two cocrystals from different mixtures of 2 and 1, including ones containing an excess of 1.The four molecules of unencapsulated ups Fig. . Analysi2 in the crystals of (22⊂1)⋅2 and (22⊂1)⋅24, the 22⊂1 units appeared nearly identical  the signals due to 2’s α protons are significantly (by ~3.0 ppm) more upfield-shifted than those of 2β and 2γ  the equatorial acidic-imidazole protons of 1 , 2’s signals became so broad that they were practically invisible. Further addition of the cage caused the reemergence and gradual sharpening of 2’s peaks; no further changes to these peaks were seen beyond ~0.5 equiv of 1 , with a strong absorption centered at λmax = 571 nm. Upon the gradual addition of 1, the intensity of this peak decreased, concomitant with an increase of a less intense, blue-shifted band at λmax = 542 nm. These changes are characteristic of the formation of an H-aggregate , as observed previously for a series of BODIPY dyes41. Notably, the 571 nm peak stopped decreasing upon the addition of 0.5 equiv of the cage, indicating that free 2 had been consumed  a strongly cooperative binding of 2 to 1, with the 2:1 complex-forming preferentially42 to a putative 1:1 complex43 .Third, the 2:1 stoichiometry is also evident from the UV-Vis and fluorescence titrations of aqueous solutions of med Fig. . This re nm Fig. . We obseded Fig. , f. This1 to a solution of 2 (solubilized in water using 1 equiv of TMEDA). By integrating the raw data, we confirmed the 2:1 binding stoichiometry  × 105 m−1 and Ka2 = 4.0(±0.4) × 106 m−1, i.e., in the range of Ka values typical of homo-49 and heteroternary53 inclusion complexes of cucurbit[8]uril and other large macrocyclic hosts. The binding cooperativity can be expressed quantitatively in terms of an interaction parameter α (= 4Ka2/Ka1 for a 2:1 stoichiometry)55 and the Hill coefficient56, nH = 2/[1 + (Ka1/Ka2)1/2]. The high values of α ≈ 57 and nH ≈ 1.58 confirm the strongly cooperative nature of the binding of 2 within 1 . The facile formation of a 2:1 complex can be understood by analyzing the crystal structures of 22⊂1, which show  experiments, in which we gradually added cage how Fig.  that the57. Curve fitting with the 2:1 binding model afforded Ka1 = 7.2(±2.4) × 105 m−1 and Ka2 = 1.5(±0.2) ×107 m−1, i.e., values slightly (by a factor of ~2.5) higher than those obtained from ITC titrations. One reason behind this discrepancy can be the large difference in the concentration of 1 in the fluorescence vs. ITC titration experiments , which affects the equilibrium between free 2 and its inclusion complexes 2⊂1 and 22⊂1. We also point out that the values derived from the fluorescence titration experiments bear some uncertainty as the emission of the putative 2⊂1 species is unknown. Nevertheless, this analysis supports the cooperative nature of the binding of 2 within 1 (Ka2 ≫ Ka1), suggesting that the initial binding of one molecule of 2 is readily followed by encapsulation of another one. It is interesting to point out an analogy to our previous study, in which we found that the formation of 2:1 complexes comprising cage 1 and two compact azobenzenes proceeds significantly faster than the formation of 1:1 complexes of the same cage and a bulkier azobenzene39.We also attempted to determine the association constants by analyzing the fluorescence titration data using the online fitting tool BindFit22⊂1 complex by the reverse-titration method, whereby a solution of cage 1 (c1 ≈ 10 mm) is titrated with guest 2. Figure 1H NMR spectra of 1 in the presence of increasing amounts of 2. Notably, since 1 is present in excess with respect to 2 at all stages of the titration, there are no free guests in the system and the signals in the spectra remain sharp throughout the experiment  shuttling of 2 between the cages .Next, we studied the assembly of the 22⊂1 was also followed by UV-Vis and fluorescence titrations . Figure 1 in the presence of increasing amounts of 2. Since all of the titrant added at the early stages of the titration is encapsulated, the ratio of Abs571nm (high for 2) to Abs542nm (high for 22⊂1) is roughly constant, and it is expected to increase only beyond ~2 equiv of the guest added. Surprisingly, however, we repeatedly observed that the ratio Abs571nm/Abs542nm increased after only 1–1.5 equiv of 2 were added , however, it was observed at an earlier point ; thus, diluting the solution shifts the complexation equilibria towards the free metal ions and ligands. Indeed, we observed that diluting a typical NMR sample of 1 (c ≈ 8 mm) by a factor of 160 (down to ~0.05 mm) not only decreases the signal intensity, but it also greatly increases the complexity of the spectra, with many new signals appearing 2(TImB)2]4+, composed of two TImB panels “stapled” by two Pd2+ ions ]2+ are occupied by water molecules; counterions = NO3−). Interestingly, however, the addition of 2 during the titration experiments can induce the reassembly of 1,At a low concentration, the equilibrium is shifted towards disassembled 1 with 2, while monitoring the fluorescence. During the initial stages of the titration, the fluorescence remained weak due to the rapid encapsulation of the fluorescent titrant . The titration was halted and the solution was allowed to equilibrate; interestingly, the emission slowly (within a few hours) disappeared, indicating that 2-templated reassembly of cage 1  took place. The oscillations in fluorescence could be repeated for multiple cycles (we followed this process during NMR titration), but only when <2 equiv of the guest were present; at >2 equiv of 2, all the cages were reassembled and the fluorescence only increased ]2+(NO3−)2  . Mass spectrometry analysis with electrospray ionization and negative ion detection afforded the expected molecular mass  of TMEDA, which preferentially sequestered the axial Pd2+ centers, disassembling the cage into 33 is further decomposed as a result of the equatorial Pd2+ reacting with TMEDA and/or OH−). The enhanced reactivity at the axial locations is noteworthy, given that both the axial and the equatorial Pd2+ have the same immediate coordination environment (one TMEDA and two TImB ligands). We note, however, that whereas the cleavage of an imidazolyl–Pdaxial bond is akin to opening a macrocyclic ring, the dissociation of an imidazolyl–Pdequatorial bond retains the overall architecture of the cage, resembling the chelate effect on a larger scale.To further confirm the relationship between 3 with 1 equiv of [Pd(TMEDA)]2+ and observed an efficient formation of 1 ⋅2, we observed the tendency of 42⊂1 to form crystals with a stoichiometry of (42⊂1)⋅4 ⋅4 and 42⊂1 were nearly identical , compared with 22⊂1 , correlates with the higher aqueous solubility of 4 compared with that of 2.Analogously to ⋅4 Fig. ; these cd 4 Fig. . The str2, resazurin 4 has an extra oxygen atom located at the center of the molecule; to accommodate the extra steric demand, the host undergoes a more significant structural distortion, developing two “compartments”, each accommodating one molecule of the guest . We found that the binding strength of both guests within 1 is comparable, in agreement with their similar molecular shapes and sizes. The binding of the first molecule of 4 (Ka1 = 1.0(±0.4) × 105 m−1) is slightly weaker than for 2, which could be due to the higher solubility of free 4. In contrast, the binding of the second guest (Ka2 = 1.2(±0.2) × 107 m−1) was stronger than for 2, which can be explained by more efficient filling of the cage’s cavity by the slightly larger 4 , whereas the overall stabilities of both 2:1 complexes are nearly the same . The association constants and thermodynamic parameters for the binding of cage 1 to both guests are listed in Table 42⊂1 is provided in Supplementary Figs. The association constants of 42⊂1 into 22⊂1. A widely used system for converting 4 into 2 is based on hydroxylamine as the reducing agent and gold nanoparticles (NPs) under green light as the photocatalyst66. In this system, green light excites the localized surface plasmon resonance band of Au NPs68, facilitating the electron transfer from NH2OH to 4. The reduction of 4 into 2 has also been achieved using triethylamine under otherwise the same conditions —a finding63 that inspired us to consider the use of TMEDA as the reductant. Here, we note that each molecule of cage 1 contains six molecules of TMEDA—also a tertiary amine—as the capping ligands on the Pd2+ nodes; we therefore hypothesized that an elegant  way to accomplish the reduction of 4 would be to use an extra amount of TMEDA . When a dilute, blue solution of 4 containing 4 equiv of TMEDA and 4.3 × 10−7 equiv of citrate-capped 21 nm Au NPs was exposed to green LED irradiation for only 12 s, it turned pink, and a UV–Vis absorption spectrum identical to that of 2 was obtained —although it was somewhat slower, most likely because of an increased steric hindrance  in the dark and in the absence of NPs and found that only ~10% of 4 was reduced to 2 overnight  over the initial 9 min of irradiation . The left-hand side of Fig. 4 and the production of 2; when plotted on a log scale, the concentration of 4 decreased linearly, indicating a pseudo-first-order reaction, with a rate constant k1 = 17.5 ± 2.6 min−1. Interestingly, once the absorption due to 2 stabilized at ~10 s, it started to decay when photoirradiation was continued significantly longer ; representative examples are shown in Supplementary Figs. The experimentally observed absorption changes were converted into concentration vs. time profiles using the Lambert–Beer law . Upon prolonged irradiation, further reduction to 52⊂1 was observed, with the solution turning colorless. The first-order reaction rate constant for the second step (k2′ = 0.057 ± 0.013 min−1) was once again much slower (by a factor of ~15.5) compared with free 2 → 5. Although 52⊂1 was too challenging to characterize, owing to its rapid oxidation back to 42⊂1, we demonstrated the ability of 1 to form a 2:1 inclusion complex with a more stable analog of 5, namely, phenoxazine 6, which lacks the OH substituents and thus is less prone to oxidation . Instead, the reaction most likely occurs on a small fraction of free 4/2 in equilibrium with the respective inclusion complex, with its rate determined by the kinetics of the unbinding of 4 and 2 from the cage. Let us consider two extreme cases: first, if the binding/unbinding events were infinitesimally slow, the reduction deceleration factor would correspond to the ratio of the equilibrium concentrations of encapsulated/free species, i.e., ~104. If, on the other hand, the kinetics of encapsulation/release were extremely fast (relative to the rate of reduction), there would be no difference between the reduction rate in the presence or the absence of the cage. Our finding that the reactions slowed down by factors of ~25 and ~15.5 indicate fast binding/unbinding of guests within/from cage 1  – in agreement with the open structure  of the cage. To confirm this reasoning, we repeated the reaction 42⊂1 → 22⊂1  in the presence of increasing amounts of a free cage . The extra cage decreases the availability of free 4 and it is expected to further decelerate the reaction. Indeed, we found that the reaction rate was inversely proportional to the amount of 1 in the system ⊂1 . If the stability of the two homodimers was significantly higher than that of (2⋅4)⊂1, or if the kinetics of guest exchange were slow, the resulting spectrum would correspond to a superposition of the two homodimer spectra; however, we observed the emergence of a distinct absorption band centered at 556 nm, which can be attributed to the heterodimer ⊂1 exists in equilibrium with the two homodimers. Interestingly, the modified spectrum was observed instantly after mixing 22⊂1 and 42⊂1, confirming the rapid kinetics of guest exchange. Second, we found that the 1H NMR spectrum obtained after mixing 22⊂1 and 42⊂1 was more complex than the sum of the spectra of the two homodimers; in addition to two sets of three signals , we observed a new set of six signals, which can be attributed to the guests within (2⋅4)⊂1 ⊂1—diffused at approximately the same rate, as expected ⊂1; it also indicates that the proximity of 4 quenches the emission of 2 in a way similar to that observed within the 22⊂1 homodimer.We note that since each cage molecule is filled with two guests, the reduction of )⊂1 Fig. , bottom.1 modulates the reduction kinetics of 4 to 2 and then 2 to 5. To this end, we painted a blue, eight-petal flower on vellum paper soaked with TMEDA and a minute amount of Au NPs (far below the detection limit of the human eye). Although the flower appeared uniformly blue, its petals were painted using free and encapsulated 4, in an alternating fashion . During the same time, petals “B” turned purple owing to the partial reduction of 42⊂1 to 22⊂1  of both dyes, dramatically changing their optical properties. In particular, we found that the strong emission of free resorufin is effectively quenched. Although the cage was previously shown to form 1:2 complexes with various flat, aromatic molecules41, only the use of water-soluble guests reported here allowed us to explore the cooperative nature of binding within 1, which we confirmed independently by ITC and fluorescence titration experiments. We also observed contrasting behaviors in host-to-guest vs. guest-to-host titrations and attributed them to the disassembly of the cage at concentrations typical for UV–Vis and fluorescence spectroscopy. Remarkably, at these low concentrations, nearly half of the cage undergoes disassembly into species incapable of forming inclusion complexes. However, the addition of strongly binding guests shifted the equilibrium back to the assembled cage, demonstrating the guests’ ability to template the formation of the cage72. These results can be described by the effect of the template on lowering the cage’s critical self-assembly concentration74. We found that the encapsulation of resazurin within the cage decreased the rate of reduction to resorufin by a factor of ~25 and that the subsequent reduction to dihydroresorufin proceeded ~15 times slower. These results indicate that the cage can protect the guest against chemical transformation, analogously to the previously reported stabilization of radical initiators75, photochromic compounds76, and other sensitive species77. The limited degree of protection is in agreement with the open structure of cage 1, which results in the fast kinetics of guest exchange. Overall, our results have the potential to expand the versatility of molecular probes for following redox processes in biologically relevant environments through the use of supramolecular hosts.In sum, we investigated the supramolecular complexation of the resazurin and resorufin dyes by a Pd1 was synthesized according to ref. 76. Briefly, TMEDA  was dissolved in 6 mL of DMSO and Pd(NO3)2·xH2O  was added. The mixture was heated at 80 °C until Pd(NO3)2 dissolved (~10 min). Next, TImB  was added, and the mixture was heated for an additional 2 h. The solution was then filtered through cotton wool to remove a dark flocculate, and 25 mL of ethyl acetate was added to the filtrate. The resulting precipitate was centrifuged, washed 4–5 times with anhydrous acetone, and dried under vacuum, resulting in a white powder.All commercial chemicals were used as received unless stated otherwise. Cage 2O or DMSO-d6. 1H NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer, a Bruker Avance III HD 500 MHz spectrometer, and a Bruker Avance III 600 MHz spectrometer. 13C NMR spectra and all 2D NMR spectra  were recorded on a Bruker Avance III 600 MHz spectrometer. Chemical shifts (δ) are expressed in parts-per-million (ppm) and reported relative to the resonance of residual solvent . For 1H NMR spectra recorded at high dilution, a water suppression program was used.NMR spectra were recorded in DUV–Vis absorption spectra were recorded on an Agilent Cary 60 spectrophotometer using quartz cuvettes with 1 and 0.4 cm pathways. Emission and excitation spectra were recorded on a Shimadzu RF-5301 PC spectrofluorophotometer.T = 25 °C). Stock solutions of 1 (2.34 mm), 2 , and 4 (0.44 mm) in double-distilled water were prepared . The syringe was charged with 100 μL of the stock solution of 1 and the cell was charged with 300 μL of the stock solution of 2 or with 300 μL of the stock solution of 4. In both titration experiments, 39 aliquots of the stock solution of 1 were injected; the first aliquot was 0.4 μL; the remaining 38 were 1 μL each. Each injection was carried out over 4 s, with the injections separated by 150 s. The stirring rate was 750 rpm, the feedback was set to high, and the reference power was 10 μW. The blanks (stock solution of 1 added to pure water) were also recorded and then subtracted from the titration data with the point-to-point tool. Subtracting the second blank  did not affect the quality of the fit. Data analysis was performed using MicroCal PEAQ-ITC Analysis Software; the Two Sets of Sites model was used to estimate the binding constants. The results are shown in Supplementary Figs. 2 and 4, respectively.ITC experiments were performed on a MicroCal PEAQ-ITC instrument under ambient conditions  was prepared by dissolving 40 mg of HAuCl4·3H2O in 4 mL of water. Gold seeds were obtained by injecting, under vigorous stirring, 0.5 mL of the gold(III) stock solution into 75 mL of a boiling water solution containing 42 mg of sodium citrate. After 10 min, the mixture was cooled down to 90 °C. Then, 0.5 mL of 60 mm sodium citrate solution and 0.5 mL of gold(III) stock solution were sequentially injected under continuous stirring, and the reaction mixture was kept at 90 °C for 30 min. Then, the injection of the same sodium citrate and gold(III) solutions were repeated and the mixture was stirred at 90 °C for an additional 30 min. Finally, the mixture was cooled down to room temperature by placing it in a water bath. The obtained NPs had a diameter of 21.2 (±3.2) nm  was dissolved in 4 mL of water. A 160 nmol of TMEDA and 9.0 × 10−7 nmol of citrate-capped Au NPs  were added. The vial was purged with argon, tightly sealed, and exposed to a green LED. The progress of the reaction was monitored by UV–Vis absorption spectroscopy. For photoreduction of 2/4 on a paper  (collimated LED power of 900 mW). Double-distilled, degassed water was used as the solvent in all photoreduction experiments. Water was degassed by repeated freeze-pump-thaw cycles. In a typical experiment, 40 nmol per Fig. , a pictu22⊂1)⋅2, 3, and 62⊂1 were obtained by slow evaporation of water from the aqueous solutions of 22⊂1, 3, and 62⊂1, respectively. Single crystals of (22⊂1)⋅24 were obtained by slow diffusion of acetone vapors into an aqueous solution of 22⊂1 in the presence of several equivalents of TMEDA. Single crystals of 42⊂1 were obtained by slow diffusion of acetone vapors into an aqueous solution of 42⊂1 in a refrigerator. Single crystals of (42⊂1)⋅4 were obtained by slow diffusion of acetone layered on top of an aqueous solution of 42⊂1 in a refrigerator. Data collection was performed under a stream of nitrogen at 100 K using a Rigaku XtaLAB AFC12 (RINC) diffractometer or a Rigaku XtaLAB Synergy-R diffractometer using Cu-Kα radiation (1.54184 Å)  and processed with CrysAlisPRO. The structures were solved by direct methods using SHELXT. All non-hydrogen atoms were further refined by SHELXL80. The crystal of 62⊂1 was twinned and was treated as such. The SQUEEZE procedure81, implemented into PLATON82 or OLEX283, was applied for disordered lattice solvent molecules, one heavily disordered nitrate in (22⊂1)⋅24, and two nitrates in 3. Crystals of 3 were particularly unstable outside the mother liquor; thus, the single crystal was directly transferred to a diffractometer and frozen with the mother liquor . The single crystal was twinned, but the separation of a single component did not improve the results. The crystal data and the structure refinement data are summarized in Supplementary Table Single crystals of 22⊂⋅2, 3, anSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


This nanosized [4+4] cage  has shape of a truncated tetrahedron with four baskets at its vertices and four aromatic amines forming the faces. Importantly, tris‐aldehyde basket 2 and aliphatic tris‐amine 7 undergo condensation to give small [1+1] cage 6. The imine metathesis of 6 and aromatic tris‐amine 4 into CBC 5 was optimized to bias the equilibrium favouring 6. Addition of tribromoacetic acid (TBA) as a chemical fuel perturbs this equilibrium to result in the transient formation of CBC 5, with subsequent consumption of TBA via decarboxylation driving the system back to the starting state.Living systems use chemical fuels to transiently assemble functional structures. As a step toward constructing abiotic mimics of such structures, we herein describe dissipative formation of covalent basket cage CBC  Dynamic covalent basket cages can now be made to exist for a tunable period of time as a function of the chemical fuel used to drive their formation. The stage is thus set to explore the functional characteristics of these fascinating molecules and mimic the action of natural systems capable of directing folding, catalysis, and transport in a spatiotemporal manner. Porous organic cages (POCs)tris‐aldehyde basket 2,tris‐(4‐aminophenyl)benzene 3 to give covalent basket cage 1  is a truncated [4+4] tetrahedron1 and developing a method for their transient formation via consumption of chemical fuel will set the stage for examining temporal control of their action,Emergence of complexity in natural systems arises from vast networks of dynamic assembliestris‐aldehyde 2 to tris‐amine 3 , it appeared that 1,2‐dichloroethane (DCE) and chloroform would, in the presence of catalytic TFA,1. Despite such optimization, cage 1 precipitated from the solution as a pale‐yellow solid being sparingly soluble in organic media. To address the issue, we prepared tris‐amine 4  appended to the benzene core. In DCE containing catalytic TFA, the reaction of tris‐aldehyde basket 2 and tris‐amine 4 resulted in the formation of CBC 5 as the sole product  with resonances arising from the basket and linker components in equal ratio. A greater magnetic deshielding of HG/H signals from tris‐imine panels is in line with the conversion of the amine functional groups into more electron withdrawing imines. From the 2D NOESY spectrum of 5 , we noted cross peaks between HF and HG as well as HF and CH2 from hexyl groups to corroborate the proximity of the two building blocks and E configuration of the imine double bond  therefore corroborating that the amine and aldehyde components are within the same molecule.After adding 3 Figure D in DMSOtris‐aldehyde basket 2 gave single crystals. After being subjected to X‐ray diffraction analysis .sp2−H groups to form a seam of edge‐to‐face Csp2−H⋅⋅⋅π hydrogen bonds at the equator  residing in the cavity of 2 and the known propensity of baskets to trap haloalkanes in solution,5 could use its four compartments for complexing complementary and polarizable CBr4 (108 Å3).4 to tetravalent CBC 5 caused a notable magnetic deshielding of its aromatic HC protons  was more favorable than by basket 2 . We posit that a more rigid and preorganized cavities of CBC 5  solution of 5 containing CBr4 resulted in the formation of single crystals. X‐ray diffraction analysis of the sample revealed rigid CBC 5 with the shape of a truncated tetrahedron having four molecular baskets at its corners conjugated to four trivalent aromatic panels by imine bonds 4⊂5] complexes . Subsequent addition of a base (Et3N) resulted in the deprotonation of [4‐H3]3+, followed by the exclusive reformation of CBC 5. We recognized that the pH‐dependent manipulation of this equilibrium could allow us to transiently form CBC 5 using an acid that dissipates over time. cage 6 under thermodynamic control. The solid‐state structure of 6 .46 Size‐5 and [1+1] cage 6 use tris‐aldehyde 2 as a subunit, we hypothesized that combining tris‐amine 4 and tris‐amine 7 in the same reaction would result in an equilibrium between the two cages under TFA catalysis . Importantly, a greater quantity of TFA shifted equilibrium III to the left with the removal of 6 and the formation of CBC 5 . As the amount of TFA tunes the state of equilibrium III in solution, we turned to probe the action of labile tribromoacetic acid  hoping that it would provide the same level of control. That is to say, an excess of TBA was expected to disturb equilibrium III  thereby triggering the removal of the smaller [1+1] cage 6. Condensation of the released tris‐aldehyde basket 2 and aromatic amine 4 (pKa=4) should then result in the transient formation of CBC 5. The tribromoacetate ion formed through protonation of 7 undergoes thermal decarboxylation to give CHBr3 and CO2 (waste) with the overall loss of acid in solution. The resulting deprotonation of aliphatic tris‐amine 7 restores the original equilibrium III dominated by aromatic tris‐amine 4 and [1+1] cage 6. In order to build a larger quantity of CBC 5 in solution, a faster degradation of 6 followed by slower dissipation of 5 must take place during the proposed reaction cycle  stayed as a precipitate  with CO2 and CHBr3 formed as waste. The process was successively repeated three times using the same reaction mixture with the concentration of [1+1] cage 6 staying consistent at 0.24–0.28 mM at the end of each cycle . Since only 60–80 % of the fuel was spent during each cycle, we had to adjust the amount of TBA to study its recyclability. As a result of this caveat and the kinetics of the reaction being highly responsive to the amount of acid, we noted that the quantity of CBC 5 dropped from cycle 1 to 3 at the 5 h mark . As recyclability is essential for the application of these systems in the future, the apparent, and still unclear, drop in the effectiveness needs to be further investigated; in this regard, the knowledge of the reactions’ kinetics is necessary for understanding and optimizing the outcome of coupled chemical processes, which we herein completed by using trial‐and‐error method. By using different quantities of TBA fuel at the beginning of the experiment, we found that the formation of CBC 5 could be adjusted to peak at 1.7, 4.5 and 12 h  can now be made to exist for a period of time,The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. 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The interaction of metallodrugs with proteins influences their mechanism of action and side effects. In the case of platinum drugs, copper transporters modulate sensitivity and resistance to these anticancer agents. To deepen the knowledge of the structural properties underlying the reactivity of platinum drugs with copper transporters, we studied the interaction of kiteplatin and two of its derivatives with the methionine‐rich motif of copper importer Ctr1 and with the dithiol motif of the first domain of Menkes ATPase. Furthermore, cellular uptake and cytotoxicity of the three complexes were evaluated in cisplatin‐sensitive and ‐resistant ovarian cancer cells, comparing the data with those of clinically relevant drugs. Reactivity depends on the tightness of the chelate ring formed by the carrier ligands and the nature of the leaving and entering groups. The results highlight the importance of subtle changes in the platinum coordination sphere that affect drug absorption and intracellular fate. Kiteplatin, a promising anticancer drug that overcomes resistance to cisplatin, binds to a methionine motif of Cu importer Ctr1 and to a dithiol motif of the first domain of Menkes ATPase, which can modulate drug uptake and intracellular distribution. The interaction is influenced by the type of incoming ligands, the nature of the leaving groups (two chlorido ligands) and the chelating character of the carrier ligand . The discovery of cisplatin has revolutionized the treatment of certain types of tumors, like ovarian, testicular and lung cancer.1, 2 TR,2R‐diaminocyclohexane  as carrier ligand, and kiteplatin, [PtCl2]  drugs capable to overcome cisplatin resistance either acquired during cycles of therapy or intrinsic .lohexane R,2R‐DACHExperimental evidences support the role of copper (Cu) transporters in the uptake of and resistance to cisplatin and its derivatives, confirming the tight connection between Cu homeostasis and cellular response to Pt‐drugs.8, 9The high‐affinity Cu influx transporter Ctr1 transfers Cu(I) ions across the plasma membrane to distinct cytosolic soluble chaperones, which ultimately supply Cu to cuproenzymes.13, 14Human Ctr1 (hCtr1) is a glycosylated protein of 190 amino acids, which consists of three putative transmembrane (TM) segments,In addition to the downregulation of Ctr1, an upregulation of two Cu pumps, ATP7A and ATP7B, may also play a key role in resistance to Pt‐based drugs.20, 21ATP7A is a Cu‐exporting P‐type ATPase which regulates the cytoplasmic Cu concentration; in particular, it is involved in the transport of Cu(I) through the secretory pathway and in Cu(I) excretion across the plasma membrane.N‐terminal domain of ATP7A  was incubated with cisplatin or oxaliplatin, the protein was able to bind Pt(II) in a bidentate manner, with the two‐cysteine (Cys) motif CxxC replacing the chlorido ligands and the oxalate group, respectively. (cbdca)] (oxa)]  was formerly tested in the pair of ovarian cancer cell lines A2780/A2780cisR (sensitive/resistant to cisplatin) where the complex showed IC50 values  of 0.8 and 4.0 μM, respectively, resulting more potent than cisplatin, which showed IC50 values equal to 2.7 and 21.6 μM, respectively. Moreover, kiteplatin was able to partially overcome cisplatin resistance, as evidenced by the resistance factor  of 5.0 to be compared with that of cisplatin (RF=8.0). Since Pt accumulation is critical for the cytotoxicity of a Pt drug, cellular accumulation of kiteplatin in the A2780 cell line was evaluated by measuring the Pt content after 4 h (4.7 pmol Pt/106 cells) and 24 h (38 pmol Pt/106 cells), resulting ca. 1.3–2.0 higher than that of cisplatin.The 1 was, on average, slightly more effective than oxaliplatin and much more effective than cisplatin. In particular, it resulted more effective than cisplatin by a factor of 1.1–5.7. In addition, complex 1 was able to overcome resistance to cisplatin while complexes 3 and 4 were not.2+   at 25 °C and the reaction course monitored by ESI‐MS  ion loses the carrier ligands  and one or two chlorido ligands.2) was incubated with Mets7, the ESI‐MS spectrum recorded after 24 h of incubation showed the binding of {Pt}2+ to the peptide  and triply charged species (m/z 398.3). The intensity of these peaks increased after 48 h of incubation with the concomitant decrease in intensity of the apopeptide peak. Moreover, the spectrum showed a low intense peak at m/z 500.2 corresponding to triply charged species containing two {Pt}2+ moieties bound to the peptide .Summarizing this part, the ESI‐MS spectra indicate that, while the 1,4‐DACH ligand of kiteplatin is displaced by Mets7, 1cis‐1,4‐DACH)(oxa)]) (3) and the second having a cyclobutanedicarboxylate (cbdca) leaving group (cbdca)]) (4). The ESI‐MS spectrum obtained after 24 h of incubation of Mets7 with 3 showed that the predominant peak was that of the apopeptide m/z 884.5  with Mets when the chelating oxa ligand was present as a leaving group instead of two chlorido ligands.The peptide was also incubated with two derivatives of kiteplatin, the first having an oxalate (oxa) leaving group ([Pt(oxa)])  and the )(oxa)])  and the 3, there was no significant reaction between 4 (the cbdca derivative) and Mets7 after 48 h of reaction  ion  is even more evident. Conversely, when Mets7 reacted with oxaliplatin, the effect of metal binding to the peptide is moderate, as evidenced by the reduction of the band at 195 nm, indicating only a slight conformational change of the peptide  displayed a negative band at 195 nm, which is characteristic of an unstructured peptide. Addition of one equivalent of kiteplatin produced a conformational change after 24 h of incubation with the appearance of a positive band at 200 nm, corresponding to a β‐turn‐type structure  and 1196.1 (7+ charge), corresponding to the apoprotein  and 1239.7 (7+ charge) appeared, corresponding to the adduct with the {Pt(DACH)}2+ moiety ‐binding site composed of two closely spaced cysteines. The CxxC motif is also the preferred binding site of Pt(II) drugs. Mnk1 was shown to coordinate to cisplatin and oxaliplatin through Cys residues, with the Pt ion retaining the ammine ligands.3 and 4), the reactions were carried out under the same conditions described above. Sample aliquots were 3‐fold diluted and analyzed by ESI‐MS at time zero and after 24 h and 48 h incubation. The ESI‐MS spectra recorded in the reaction with complex 3  are reported in Figure m/z=1252.6 and 1239.7 (7+ charge), corresponding to mono‐ and bidentate adducts of Mnk1 with {Pt(DACH)(oxa)} and {Pt(DACH)}2+ respectively, increased in intensity. After 48 h of reaction, the peaks corresponding to the monodentate adduct (i. e. Mnk1 binding to {Pt(DACH)(oxa)} through only one Cys) were no longer detectable due to the loss of the oxalate group, but a residual amount of apoMnk1 was still present.With the two derivatives of kiteplatin .No binding of Pt to the protein was observed when Mnk1 was reacted with complex 1, 3, and 4 to overcome cisplatin resistance. Further studies concerning the reactivity toward intracellular nucleophiles or the involvement of other transporters, such as OCTs, are necessary to elucidate the different mechanism of action that makes kiteplatin so different from its derivatives.Since the interaction of Pt‐based drugs with copper transport proteins deeply influences their cellular accumulation and hence their activity, the present investigation is a first step in the understanding of the different ability of complexes cis‐1,4‐DACH as well as one or two chlorido ligands, and the Pt(II) ion binds to the peptide as occurs with cisplatin. The oxalate derivative 3 exhibits the same behavior, albeit with a slower reaction rate. On the other hand, oxaliplatin retains the 1R,2R‐DACH ligand after 48 h of reaction. Oxaliplatin and complex 3 have the same molecular formula but a different spatial position of the −NH2 groups on the DACH carrier ligand. This change could explain the different reactivity of 3 and oxaliplatin toward Mets7. The 1R,2R‐DACH ligand forms a stable 5‐membered chelate ring with Pt(II), while 1,4‐DACH forms a loose 7‐membered ring. The least reactive compound is complex 4; in the ESI‐MS spectrum recorded after 48 h no peak corresponding to an adduct with the peptide was observed, probably due to the steric hindrance of the cbdca leaving group.In the present work, using the ESI‐MS and CD spectra, we demonstrated that when the Mets7 peptide is incubated with kiteplatin, the latter loses the carrier ligand 3 and 4: while the oxalate group in complex 3 is displaced by Mnk1 }2+ moiety), the cbdca ligand in complex 4 hinders the coordination of the protein.Kiteplatin and oxaliplatin have similar reactivity toward the first domain of ATP7A, as they maintain the DACH group and lose the chlorido and oxa ligands, respectively. The effect of the leaving group is evident in the reaction of Mnk1 with Synthesis of Pt complexes. [PtCl2] ,R,2R‐DACH)(oxa)] ,cis‐1,4‐DACH)(cbdca)] (4)cis‐1,4‐DACH)(oxa)] (3): oxalic acid  was dissolved in water (40 mL) and the resulting solution was treated in the dark with Ag2O  for 15 min at 50 °C. The resulting suspension was added to 1 , stirred with a magnetic stirrer in the dark at 50 °C for 90 min and then at room temperature for 24 h. The white precipitate that formed (AgCl) was filtered through celite and the filtrate evaporated under vacuum at 40 °C. The light yellow residue  corresponds to [Pt(oxa)].+ [3+Na]+: 420.0. Found: m/z 420.0. Anal.: calculated for C8H16N2O5Pt (3 ⋅ H2O): C, 23.14; H, 3.88; N, 6.74 %. Found: C, 23.44; H, 3.61; N, 6.73 %.Cytotoxicity assays. The number of living cells was evaluated by the 3‐‐2,5‐diphenyltetrazolium bromide (MTT) assay. Briefly, A2780 human ovarian endometrioid adenocarcinoma cells and cisplatin‐resistant A2780/Cp8 ovarian cancer cells were plated in 96‐well plates at a density of ∼5000 cells/well. After one day incubation at 37 °C in a humid atmosphere with 5 % CO2, the culture medium was replaced with 100 μL of fresh medium (control cells) or medium containing different concentrations of the test compounds in the range 0.125–50 μM. Untreated cells were used as positive control. After the incubation period of 72 h, 10 μL of a 0.5 % (w/v) MTT/PBS solution were added to each well and the incubation was prolonged further for 3 h. After this period, medium was removed and replaced with 100 μL of DMSO. The absorbance of the individual well was measured by a microplate reader . Each compound concentration was tested in triplicate, and results presented as percentage of the control value. IC50 values were calculated using nonlinear regression in GraphPad Prism 5.01.Sample preparation for ESI‐MS experiments. The Pt complexes were dissolved immediately prior to use in pure water at 1 mM final concentration. The complex solution was extensively vortexed and sonicated. The peptide Mets7, acetylated at its N‐terminus, was purchased from GenScript Corp. (USA). The purity was validated to be greater than 98 % by analytical HPLC and the mass was confirmed by ESI‐MS . Mets7 was dissolved in pure water at 1 mM concentration. Mnk1 was recombinantly expressed by standard biotechnological techniques, using Escherichia coli BL21‐Gold (DE3) competent cells  and pET vectors under the control of the Isopropyl‐β‐D‐1‐thiogalattopyranoside (IPTG) inducible lac promoter . Purification was achieved by immobilized metal affinity and size‐exclusion chromatography on an automated AKTA‐Purifier UPC900, equipped with a detector at 280 nm. After cleavage of the (His)6 purification tag, four amino acids (IEGR) of the restriction site were left at the C‐terminus of the protein. The N‐terminal methionine was partially processed in E. coli, yielding a 76‐amino acid protein (numbering the first codon Met as residue 1). All purification steps were carried out in the presence of an excess of the reducing agent dithiothreitol (DTT), to preserve the protein in its active form. Protein purity was determined by SDS‐PAGE and ESI‐MS . To extensively remove DTT prior to Pt‐drug addition, protein samples were washed with deoxygenated water by using Amicon Ultracentrifugal filters with 3 kDa cutoff , under N2 atmosphere, and immediately used for the incubations.Electrospray ionization mass spectrometry. ESI‐MS was performed with an electrospray interface and an ion trap mass spectrometer .v/v) was added before injection in order to obtain a good volatilization. Ionization was achieved in the positive ion mode by application of +4 kV at the entrance of the capillary; the pressure of the nebulizer gas was 15 psi. The drying gas was heated to 350 °C at a flow rate of 5 L/min. Full‐scan mass spectra were recorded in the mass/charge (m/z) range of 50–2200. In the reaction with Mnk1, an equimolar amount of 1, 3 or 4 was added to Mnk1 (100 μM) at 25 °C under anaerobic conditions. Aliquots were removed at different time intervals, 3‐fold diluted, and analyzed by ESI‐MS as previously described.The reaction between Mets7 (50 μM) and kiteplatin (50 μM) or oxaliplatin (50 μM) was carried out at 25 °C. Aliquots of the reaction mixture were removed at different time intervals after mixing and injected at a rate of 10 μL/min. 1 % acetic acid . The cells were seeded in 60 mm tissue culture dishes at a density of 500,000 cells and incubated at 37 °C in a humidified atmosphere with 5 % CO2. After overnight incubation, the culture medium was replaced with 3 mL of medium containing the compounds at 10 μM concentration and incubated for 4 h and 24 h. After the incubation period, the cell monolayer was washed twice with ice‐cold PBS. The samples were vortexed in the presence of 500 μL of 65 % HNO3  and then digested overnight at room temperature. Finally, the solutions were diluted to 10 mL with Milli‐Q water. Tune A solution  was used to tune the ICP‐MS instrument. Bismuth was used as an internal standard, hence 20 μL of its solution at 500 μg/L were added to the samples prior to analysis. All samples were analyzed three times. The isotopes monitored were 195Pt and 209Bi and the Pt concentration was determined by comparison with an external calibration curve . The detection limit was calculated as the concentration of an element that gave a signal equal to three times the standard deviation of a series of ten successive measurements of the blank solution.Circular dichroism. Far‐UV CD spectra were recorded at different time intervals on apoMets7 (50 μM) incubated with 1‐4 (50 μM) in pure water using a Jasco J‐810 spectropolarimeter , a quartz cuvette with a 1 mm optical path, a wavelength interval of 190–250 nm and 0.1 nm data pitch. All spectra, corresponding to an average of 10 scans, were baseline corrected and then smoothed by applying adjacent averaging or a fast Fourier transform filter. The ellipticity is reported as mean residue molar ellipticity (deg ⋅ cm2 ⋅ dmol−1) according to [θ]=100 [θ]obs/(C ⋅ L ⋅ N), where [θ]obs is the observed ellipticity in degrees, C is the molar concentration of the protein, L is the optical path length (in cm), and N is the number of amino acids (N=8 for Mets7).The authors declare no conflict of interest.