Rhodiola rosea, which contains a complex mixture of phytochemicals, reported some positive drug-drug interaction (DDI) findings based on in vitro CYP450’s enzyme inhibition, MAO-A and MAO-B inhibition, and preclinical pharmacokinetic studies in either rats or rabbits. However, variation in and multiplicity of constituents present in Rhodiola products is a cause for concern for accurately evaluating drug-drug interaction (DDI) risk. In this report, we examined the effects of bioengineered, nature-identical salidroside on the inhibition potential of salidroside on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 utilizing human liver microsomes, the induction potential of salidroside on CYP1A2, CYP2B6 and CYP3A4 in cryopreserved human hepatocytes, the inhibitory potential of salidroside against recombinant human MAO-A and MAO-B, and the OATP human uptake transport inhibitory potential of salidroside using transfected HEK293-OATP1B1 and OATP1B3 cells. The results demonstrate that the bioengineered salidroside at a concentration exceeding the predicted plasma concentrations of <2 µM (based on 60 mg PO) shows no risk for drug-drug interaction due to CYP450, MAO enzymes, or OATP drug transport proteins. Our current studies further support the safe use of salidroside in combination with other drugs cleared by CYP or MAO metabolism or OATP-mediated disposition.Several studies utilizing  Rhodiola rosea to increase physical endurance, work productivity, longevity, resistance to high altitude sickness, and to treat fatigue, depression, anemia, impotence, gastrointestinal ailments, infections, and nervous system disorders  ethanesulfonic acid (HEPES) were purchased from Gibco. Estradiol-17β-glucuronide was from Toronto Research Chemicals and cylcosporin A from J & K.The inhibition potential of salidroside (0.0488–50 µM) on the activities of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes were tested. Prototypical positive controls (β-naphthoflavone (0.0039–0.4 µM), ticlopidine (0.0012–1.2 µM), quercetin (0.0039–4 µM), sulfaphenazole (0.0012–1.2 µM), tranylcypromine (0.0117–12 µM), quinidine (0.0001–0.12 µM), and ketoconazole (0.0004–0.40 µM)) were included.50 values were determined by the Hill slope equation using Graph Pad Prism.Test compound/controls working solution or DMSO were added in 238.5 µL of liver microsomes working solution, mixed well by pipetting several times and pre-incubated in a 37 °C shaking water bath for 5 min. Following the pre-incubation, 60 µL of NADPH working solution were added, mixed well by pipetting several times, and incubated in a 37 °C shaking water bath for 10 min. Following the incubation, 300 µL of quenching solution were immediately added and vortexed for ~20 s to mix. All samples were centrifuged at 4000 rpm for 15 min at 4 °C and then the supernatant was transferred for LC-MS/MS analysis. IC50 shift is determined by using the equation of IC50 without 30 min preincubation/IC50 with 30 min preincubation. IC50 shift value of >1.5-fold is considered positive for TDI.The potential for time dependent inhibition (TDI) of salidroside was tested by the IC50 shift assay for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, utilizing a pool of human liver microsomes (Lot ZZQ). Typical substrates (phenacetin (50 μM), bupropion (80 μM), amodiaquine (2 μM), diclofenac (10 μM), S-mephenytoin (25 μM), dextromethorphan (5 μM), and testosterone (50 μM), respectively). A specific amount of each positive control compound  propan (PPP), and 20 mM gemfibrozil 1-O-β-glucouronide) was independently prepared as stock solutions in DMSO, then the first five stock solutions were equally mixed into a five-in-one stock solution. Incubation without 30 min pre-incubation, 178.5 µL of liver microsomes working was pre-incubated in a 37 °C water bath for 30 min. After the pre-incubation, 1.5 μL of test compound or control compound working solution, 60 μL of substrate working solution, and 60 μL of NADPH working solution were added; then, the reaction mixture was mixed by pipetting up and down and then incubated at 37 °C for 10 min. Incubation with 30 min pre-incubation. Next, 1.5 μL of test compound or control compound working solutions and 60 μL of NADPH working solution were added into 178.5 μL of liver microsome working solution, then the reaction mixture was mixed by pipetting up and down and then pre-incubated at 37 °C for 30 min. After the pre-incubation, 60 μL of substrate working solution was added, then the reaction mixture was mixed by pipetting up and down and then incubated at 37 °C for 10 min. Following the incubation, 300 μL of quenching solution was added into the corresponding wells to terminate the reaction. All the above samples were vortexed for 1 min and centrifuged at 4000 rpm, 4 °C for 15 min. After centrifugation, the supernatant was removed for further LC-MS/MS analysis. ICThe induction potential of salidroside on CYP1A2, CYP2B6, and CYP3A4 in cryopreserved human hepatocytes was tested. The four donors’ information is listed in 2. The sandwich medium was removed, and the hepatocytes were treated with incubation solutions containing salidroside , vehicle (0.1% DMSO), or positive inducers in triplicate for 24 h after the cultures were established. The incubation solution was aspirated and replaced with incubation solution containing the same concentrations of salidroside, vehicle, or positive inducers for an additional 24 h. The total treatment period was 48 h. After the treatment period of 48 h, the incubation solution was aspirated. Hepatocytes were washed twice with pre-warmed HBSS containing 10 mM HEPES (HBSS I) and then the remaining solution was aspirated. Hepatocytes were washed once with pre-warmed HBSS I and incubated with 140 μL of RLT (lysis buffer from RNeasy 96 Kit) supplemented with 1% β-mercaptoethanol. Plates were then stored at −70 °C in a freezer until RNA analysis. Before RNA extraction, the frozen plate was thawed, and cell lysates were transferred to 96-well plates. RNA isolation was performed using a QIAcube RNA extraction system. Reverse transcription was performed to obtain cDNA. Quantification of the selective genes by real-time quantitative polymerase chain reaction (qPCR) was performed with TaqMan universal PCR Master Mix on the Bio-rad CFX384 TouchTM Real-Time PCR System. The relative change in the mRNA level of the selected target genes induced by each test compound was expressed in relation to the vehicle control sample, and fold changes in gene expression were determined by the ΔΔCt method.All hepatocyte incubations were conducted at 37 °C, 95% air/5% CO50 values were determined by the Hill slope equation using Graph Pad Prism.A total of 99 µL of substrate working solution was added into 1.1 mL incubation tubes and then 1 µL of the test compound/control working solution or DMSO (vehicle control) was added, mixed well, and pre-incubated at 37 °C for 5 min. MAO-A (0.005 mg/mL) and MAO-B (0.02 mg/mL) were pre-incubated in the working solution at 37 °C for 5 min. A total of 100 µL of pre-incubated MAO-A or MAO-B working solution was added to incubation tubes to start the reaction, mixed well, and incubated at 37 °C for 15 min. Following the incubation, immediately add 200 µL of quenching solution were immediately added and vortexed to mix. All samples were centrifuged at 4000 rpm at 4 °C for 15 min, then 100 µL supernatant with 100 µL of water was transferred for LC-MS/MS analysis. IC6 cells/mL with DMEM supplemented with 1% non-essential amino acid (NEAA), 10% fetal bovine serum (FBS), 1% PS solution (100 U/mL penicillin-G and 100 μg/mL streptomycin), and seeded on a 96-well BioCoat™ poly-d-lysine plate at a density of 1 × 106 cells/mL. The cells were incubated at 37 °C, 5% CO2/95% air and saturated humidity for 3–4 h. Once the cells were anchored on the plate, the cell culture was replaced with DMEM containing 10% FBS and 1% PS solution (100 U/mL penicillin-G and 100 μg/mL streptomycin solution) and cultured one day for the uptake and inhibition studies. The cell culture medium was removed from the 96-well plate, then rinsed twice with 0.1 mL/well of pre-warmed transport buffer. The transporter cells containing pre-incubation solution were pre-incubated at 37 °C for 30 min. Then the pre-incubation solution was removed and replaced with 0.1 mL/well of dosing solution containing a substrate, respectively . Then, the transporter cells were incubated at 37 °C for 2 min. After incubation, all solutions in the cells were discarded and rinsed with 0.10 mL/well of ice-cold transport buffer (4 °C) three times. After rinsing, 0.10 mL/well of distilled water was added, and the transporter cells were lysed via three cycles of freezing and thawing of liquid N2 (−196–37 °C). A total of 50 μL of cell lysate was quenched with methanol and acetonitrile containing 100 ng/mL tolbutamide and 100 ng/mL labetalol  at a volume ratio of 1:2. All samples were shaken and centrifuged at 4000 rpm at 4 °C for 15 min and the supernatant was removed for future analysis by LC-MS/MS.HEK293-OATP1B1 and OATP1B3 cells were diluted to 1 × 10DMSO solutions of salidroside and warfarin (positive control) were spiked into human and SD rat plasma to 5 μM concentrations . The experiment was performed using equilibrium dialysis with the two compartments separated by a semi-permeable membrane. Phosphate-buffered saline (PBS) (pH 7.4) was added to one side of the membrane and the plasma solution to the other side. After equilibration (5 h at 37 °C), samples were taken from both sides of the membrane, transferred to a new plate, and were matrix-matched by the addition of an appropriate amount of PBS to the plasma samples and blank plasma to the PBS samples. Methanol/acetonitrile  containing an analytical internal standard terfenadine or tolbutamide (IS) was added to precipitate the proteins and extract salidroside and IS. Incubations were performed in duplicate. Warfarin served as a control. The fraction unbound in plasma  = Cb/Cp, where Cb represents the concentration in buffer and Cp the concentration in plasma at 5 h.The following compounds were quantitatively determined by LC-MS/MS methods.For the transporter-transfected HEK293 cells transport inhibition study, the concentrations of rosuvastatin and estradiol-17β-glucuronide were used, with tolbutamide as the internal standard and for CYP induction by enzyme activity, the metabolites acetaminophen, hydroxybupropion, and 1′-hydroxymidazolam. For MAO-A and MAO-B inhibition, 4-hydroxyquinoline with tolbutamide was used as the internal standard. Salidroside and warfarin were measured in the plasma protein binding study. The HPLC-MS system utilized a Shimadzu LC 30 series instrument and Sciex QTRAP 5500 mass spectrometer 5500 with turbo ionspray . Samples were injected onto a Kinetex 2.6 µm C18 100A column (3.0 mm × 50 mm) and quantified by reaction monitoring of metabolites and internal standard. The transporter study employed a calibration curve of spiked samples and inclusion QC samples to confirm assay performance. The MAO inhibition and plasma protein binding studies used peak area ratios (PAR) of analyte/internal standard to semi-quantitatively determine the concentration of 4-hydroxyquinoline by the LC-MS/MS method or the relative concentrations of salidroside and warfarin in the buffer and plasma aliquots.The vehicle sample was the metabolite peak area ratio in the incubation lacking salidroside as an inhibitor.For competitive and time-dependent CYP inhibition, the peak area ratios (PAR) of analyte/internal standard were used to semi-quantitatively determine the concentration of acetaminophen (CYP1A2 activity), 4’-hydroxydiclofenac (CYP2C9), 4’-hydroxymephenytoin (CYP2C19), dextrorphan (CYP2D6), 1’-hydroxymidazolam (CYP3A4), 6β-hydroxytestosterone (CYP3A4), hydroxybupropion (CYP2B6), and N-desethylamodiaquine (CYP2C8), with terfenadine as the internal standard by LC-MS/MS method. The HPLC-MS system utilized an Acquity UPLC system and Sciex API 4000 mass spectrometer in ESI positive mode, with quantitation employing MRM. Samples were injected onto an Atlantis T3 5 μM column (2.1 x 50 mm).S)-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), testosterone (CYP3A4/5), and midazolam (CYP3A4/5) were used as markers for CYP activities in human liver microsomes. The IC50 values for the positive controls were in the range for historical data  enzymes was evaluated using a mixed donor pool of human liver microsomes. The probe substrates phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), diclofenac (CYP2C9),  were all >50 µM, as summarized in When salidroside was incubated at several concentrations (0.5–50 µM), there was no measurable inhibition in marker substrate metabolic rate for any of the seven CYP isoforms tested. The ICS)-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), testosterone (CYP3A4/5), and midazolam (CYP3A4/5) were used as markers for CYP activities in human liver microsomes. Positive control inhibitors for the time-dependent inhibition were included within each assay and showed a significant shift in the IC50 values, which is consistent with the literature  of salidroside towards cytochrome P450 (CYP) enzymes was also evaluated using the same lot of human liver microsomes. The experiments measured the metabolic rates of marker substrates with or without a 30 min pre-incubation of salidroside (up to 50 µM) or positive controls (known inactivators) in the presence of NADPH. The probe substrates phenacetin (CYP1A2), bupropion (CYP2B6), amodiaquine (CYP2C8), diclofenac (CYP2C9),  was preincubated with human liver microsomes in either the presence or absence of NADPH for 30 min  at 0.1, 1.0, and 10 µM by enzyme activity. Based on the results of the preliminary study, a full study in three different lots of human hepatocytes  at concentrations up to 30 µM was evaluated based on mRNA data only.CYP1A2, CYP2B6, and CYP3A4 activities in cryopreserved human hepatocytes were quantified by measuring the formation of acetaminophen from phenacetin, hydroxybupropion from bupropion, and 1-hydroxymidazolam from midazolam. Following treatment of cryopreserved human hepatocytes prepared from donor (SNP) with the prototypical inducers 50 μM omeprazole for CYP1A2, 1000 μM phenobarbital for CYP2B6, and 10 μM rifampicin for CYP3A4/5, activities were increased 3.55-, 5.69-, and 2.79-fold of vehicle control, respectively. CYP1A2, CYP2B6, and CYP3A4 activity levels in hepatocytes treated with salidroside at the concentrations of 0.1, 1, and10 µM were not increased over the positive controls in donor (SNP) , indicatInduction of CYP1A2, CYP2B6, and CYP3A4 was observed for the known inducers rifampin, phenobarbital, and omeprazole as measured by mRNA levels. Treatment of the human hepatocytes with salidroside did not cause induction of CYP1A2, CYP2B6, and CYP3A4 mRNA levels, as summarized in 50 values of the known inhibitor leflunomide were 13.8 μM and 7.40 μM  towards MAO-A and MAO-B were greater than 50.0 µM, with a maximum % inhibitions at 50 µM of 13.3% and 22.5%, respectively  was 0.0714 µM (Estradiol-17β-glucuronide (substrate) was incubated with an OATP1B1-transfected HEK293 cell monolayer in the presence or absence of salidroside at concentrations up to 100 µM. IC.0714 µM .50 was >100 µM  was 1.50 µM (50 was estimated to be >100 µM.Rosuvastatin (substrate) was incubated with an OATP1B3-transfected HEK293 cell monolayer in the presence or absence of salidroside at concentrations up to 100 µM or the positive control rifampicin. The calculated IC 1.50 µM . The meau,plasma) were 0.985 and 0.856 in human and rat, respectively.The protein binding of salidroside was determined in human and plasma by equilibrium dialysis at a concentration of 5.0 µM. Salidroside was minimally bound in plasma, as the unbound fractions in plasma (fb) of 32.1%, the estimated oral absorption (fa) would be fa = fb/((1 − CL/Qh) × 100%) or 46%. After oral administration, the observed Cmax was 4.3 mg/mL or 14.3 µM. Based on the 12-fold higher oral dose in rat relative to humans (60 mg daily or ~1 mg/kg), the equivalent predicted human total Cmax is ~1.2 µM and the unbound Cmax is ~1.18 μM .An estimation of the maximum plasma concentration of salidroside in humans was made from the pharmacokinetics in rats after oral dosing  and the Rhodiola rosea reported a variety of beneficial effects [Rhodiola rosea extracts reported various drug-drug interactions, although causative agent(s) and clinical relevance are yet to be determined. To determine if the effects observed with Rhodiola rosea extracts are specifically due to Salidroside, we tested a biosynthesized form currently available commercially. Using experimental designs recommended by the FDA, bioengineered salidroside of high purity was without either inhibitory or inductive effects on CYPs or inhibitory effects on MAO-A, MAO-B enzymes or OATP1B1 or OATP1B3 drug transporters.People taking extracts of  effects . MarketeCytochrome P450 (CYP) metabolism is a common pathway for the modification and breakdown of marketed pharmaceuticals in the human body. Seven CYP isoforms  account for >90% of enzymatic reactions catalyzed by P450s. Regarding drug metabolism, three-quarters of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A . The cyt1/2 were observed for phenacetin, bupropion, losartan, and midazolam in the single-dose salidroside-pretreated group. While the authors concluded this reflected CYP induction [0-∞ for losartan and midazolam each decreased ~2-fold following eight daily doses of salidroside. Therefore, Rhodiola rosea could potentially affect the intracellular concentrations of drugs metabolized by CYP3A4 or CYP2C9 enzyme. However, there are important species differences in the ligand preferences of these xenobiotic-sensing receptors limiting the translation of in vivo results from the experimental animals to the humans [Salidroside was not previously studied regarding potential CYP inhibition or induction using human-derived reagents. Induction of CYP1A2, CYP2B6, CYP3A4, and CYP2C9 was inferred based on the pharmacokinetic alteration of drugs in male SD rats, using specific drugs that are selective substrates for these human CYP isoforms. Three groups of six animals were used, the control receiving no pretreatment, the second receiving 30 mg/kg salidroside daily for 8 days, and the third group received 30 mg/kg salidroside on day 8. All three groups were administered the cocktail of five drugs on day 8; 2 h after salidroside or placebo administration. Reductions in AUC and te humans .In this study, both the potential competitive and time-dependent CYP inhibition and CYP induction by salidroside were assessed over a concentration range using in vitro assays employing human-derived reagents, as recommended by the FDA . At concMonoamine oxidases (MAOs) regulate the metabolic degradation of catecholamines and serotonin in the central nervous system and peripheral tissues and inhibition can alter the breakdown of these important biomolecules. Toxicity resulting from MAO inhibition includes serotonin syndrome and hypertension ,19. InteRhodiola rosea and twelve individual, isolated components were previously tested for MAO-A and MAO-B inhibitory potency [Extraction mixtures of  potency . At a coRhodiola rosea extract at 50 mg/kg in female New Zealand rabbits on the pharmacokinetics of losartan , a drug whose clearance is mediated in part by OATP hepatic uptake and by CYP2C9 and CYP3A4 metabolism, was studied. The AUC and t1/2 of losartan were increased ~2-fold [Recent reports suggest that the rate-limiting step in the overall clearance of several major classes of therapeutics, including statins and sartans, is hepatic uptake by OATP1B1 and/or 1B3 . The pot ~2-fold . TherefoThe potential for DDI interactions altering the levels of salidroside were not evaluated due to the balanced clearance of metabolism and renal clearance of salidroside, such that a complete inhibition of either mechanism would only result in a maximum 2-fold increase. Two studies determined the PK, tissue distribution, and excretion of salidroside following IV doses in rats. The percentage recovery of an IV dose as unchanged in the urine was 53.7 and 64% . Three mRhodiola rosea extracts for activity is salidroside [Thus, because of these results, engineered salidroside would provide users a better product since one of the main ingredients touted in idroside , which aidroside .We previously reported that the bioengineered salidroside is safe and not genotoxic. In addition, the no observed adverse effect level (NOAEL) for the bioengineered salidroside is ≥2000 mg/kg/day based on the multiple oral dose rat study .Rhodiola rosea extracts or conclusions based on the in vivo effects in preclinical species. Together, the results from experimental designs recommended by the FDA indicate that bioengineered salidroside of high purity can be safely applied as an ingredient in dietary supplements, as a food ingredient, and potentially as a pharmaceutical therapeutic agent, without risk of drug interaction.These prior safety results underscore the safety of a bioengineered salidroside product, even at high doses. In this paper, the results demonstrate that the in vitro testing of salidroside at concentrations exceeding the predicted plasma concentrations of <2 µM (based on 60 mg PO) showed no risk for drug-drug interaction due to CYP450, MAO enzymes, or OATP drug transport proteins. Earlier studies with positive findings appear to be caused by other constituents of


Dynamic detection of aqueous Pd2+ by the probe RCya could be accomplished through ion competition, linear response, fluorescence-pH/time stabilities, and other optical tests. Moreover, the high selectivity, low cytotoxicity, cell permeability, and lysosome accumulation properties of RCya enabled the imaging applications on solid-state RCya–PAN composite nanofibers and in living cells. The recognition mechanism of probe RCya toward Pd2+ was further studied through simulation calculation and MS analysis.Constructed on the moiety of a lactam screw ring, a near-infrared fluorescent probe RCya for Pd Palladium, as a mature and early-used platinum-group catalyst, has a wide distribution in the soil, water, and other environmental sources. When entering the food chain through contaminated food or water and being taken into the human bodies, the food-borne intake palladium would not only accumulate in the organs but bound with thio-containing amino acids, proteins, DNA, and other related biomolecules , disruptCompared with the instrumental analyses with complex requirements on instruments and sample preparations, optical detection approaches, especially the colorimetric and fluorimetric analytical approaches ,6, demon2+ was synthesized by introducing the recognition group 2,4-dihydroxybenzaldehyde into the moiety of a lactam screw ring  in the concentration range of 0–40 μmol L−1 Pd2+  (0–40 μmol L−1) could be expressed by the following equation  = 693.3237.Probe RCya: 0.210 g, green solid; yield 48.26%. −1) of probe RCya, Pd2+, Sn2+, Cu2+, Ca2+, Mg2+, Cr3+, Cd2+, Co2+, Ba2+, Pb2+, Ag+, Fe3+, Fe2+, Ni2+, Al3+, Zn2+, Mn2+, Na+ and K+ were prepared in EtOH-H2O (V (EtOH):V (H2O) = 1:99, PBS, pH = 7.4). When used for spectroscopic tests, the stock solutions were usually diluted with EtOH-H2O (V (EtOH):V (H2O) = 1:99, PBS, pH = 7.4) to 10 μmol L−1 unless noted. All the measurements were performed at least in triplicate and averaged.Stock solutions :m (PAN) = 1:1000, DMF solution) as the matrix were prepared through the electrospinning technique. When used for nanofiber imaging, the RCya–PAN composite nanofibers were then immersed into a target ion-containing aqueous solution and imaged under ambient light or under the 365 nm ultraviolet lamp.For the detection and imaging of Pd2+ and RCya–Pd2+ in HeLa cells. HeLa cells in the exponential phase of growth were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS). After digested with 0.25% trypsin solution, cells were dispensed in 96-well cell culture clusters (200 μL per well) at a density of 2.5 × 104 cells per mL, incubated at 37 °C with 5% CO2 for 24 h. The probe RCya (100 mmol L−1 in DMSO) was then added to the 96-well plates and incubated for another 24 h to achieve a concentration gradient [−1. Subsequently, the medium was removed, the cells were washed with PBS three times and incubated with 5.0 mg mL−1 MTT solution (20 μL MTT and 180 μL medium) at 37 °C for 4 h. After that, the cells were washed with PBS three times (1 mL per well) and then dissolved in DMSO (150 μL per well). The optical intensity at 490 nm, conducted in triplicate, was recorded on a microplate spectrophotometer. Data were expressed as mean ± standard deviation (SD).Methyl Thiazolyl Tetrazolium (MTT) assay  was adopgradient  of 6.25,2. After 2 h, the growth medium was removed, and the cells were firstly washed with DMEM and incubated with 10 μmol L−1 of probe RCya for 30 min at 37 °C, then washed three times with PBS and imaged [−1 Pd2+, HeLa cells were finally incubated with RCya and Pd2+ for 30 min at 37 °C, washed three times with PBS, and imaged. In the co-staining experiments of RCya–Mito/Lyso, HeLa cells were costained with 1 μmol L−1 RCya, 1 μmol L−1 Pd2+, and 100 nmol L−1 Mito/Lyso Tracker for 30 min at 37 °C, and then washed three times with PBS and imaged.HeLa cells were cultured in 10% FBS-containing DMEM at 37 °C in the humidified atmosphere with 5% COd imaged . After s


Smaller  or larger cations  are inert. The trimers display extensive CH–O interactions between the equatorial and pseudo-equatorial hydrogens of CB[7] and the carbonyl rim of the neighboring CB[7] unit in the trimer, and a deeply nested cation between the three interacting carbonylated CB[7] rims; a counteranion is likely perched in the shallow cavity formed by the three outer walls of CB[7] in the trimer. Remarkably, a guest must occupy the cavity of CB[7] for trimerization to take place. Using a combination of semi-empirical and density functional theory techniques in conjunction with continuum solvation models, we showed that trimerization is favored in DMSO, and not in water, because the penalty for the partial desolvation of three of the six CB[7] portals upon aggregation into a trimer is less unfavorable in DMSO compared to water.Cucurbit[7]uril (CB[7]) encapsulates adamantyl and trimethylsilyl substituents of positively charged guests in dimethyl sulfoxide (DMSO). Unlike in water or deuterium oxide, addition of a selection of alkali and alkali-earth cations with van der Waals radii between 1.0 and 1.4 Å (Na Cucurbit[7]uril complexes aggregate into well-defined trimers in dimethyl sulfoxide in the presence of a selection of cations, as long as the host cavity is filled with a guest that leaves one carbonylated portal available for cation binding. To encapsulate very poorly water-soluble guests into CB[n]s, we recently showed that guests can be forced into their cavity by ball-milling a mixture of the host and guest in the solid state.12 Like Kaifer and co-workers before us,13,14 this brought us to also consider alternate, non-aqueous solvent systems that would allow encapsulation. Here we will show that CB[7] not only forms tight inclusion complexes with N-adamantyl- and N-trimethylsilylmethyl-pyridinium in DMSO-d6, but also undergoes quantitative complex trimerization in the presence of an exclusive selection of alkali and alkali-earth cations, as long as a guest occupies the cavity of CB[7]. We will present an in-depth justification for this new cluster formation.While the exquisite recognition properties of CB in DMSO using a 1 : 1 binding model . In contrast, guest encapsulation in deuterium oxide affords complex CB[7]·1 quantitatively, and addition of at least 60 vol% D2O to the DMSO solution annihilates the minor assembly. Recording the 1H NMR spectrum of the DMSO sample at 100 °C did not result in any coalescence between both assemblies, thereby indicating a very slow  exchange process.Adamantyl signals Hl see ESI. The appTo make it even more enigmatic, only guests 1, 2 and 4 afforded this assembly, although all five structures 1–5 were clearly complexed with CB[7] in DMSO. The fact that these three guests are forming the same type of complex  implies that some specific steric requirements must be fulfilled to access this mysterious species.18–22 we then suspected that aggregation might take place and result in the formation of larger clusters of complex CB[7]·1 in DMSO. Bardelang and coworkers showed that in the presence of sodium cations (≥5.0 mM), CB[8]-bound nitroxide radicals undergo trimerization in D2O; they also observed a small amount of aggregation in the absence of added sodium, and attribute it to the trimer without its sodium core cation.19Inspired by the work of Bardelang and coworkers,2O even in the presence of sodium chloride, addition of sodium cation to a DMSO solution of complex CB[7]·1 triggered its conversion to the other unknown assembly; quantitative conversion was observed in the presence of 2.3 mM Na+. Again, addition of at least 60% D2O quantitatively restored complex CB[7]·1. Diffusion-ordered spectroscopy (DOSY) of a mixture of both assemblies afforded diffusion coefficients D of 9.7 (±0.1) × 10−11 and 6.9 (±0.1) × 10−11 m2 s−1 for complex CB[7]·1 and the unknown assembly, respectively (log D −10.02 (±0.01) and −10.16 (±0.01), respectively). Power law M being the molecular weight of the analyte,23–25 can be rearranged into M); m ranges from approximately 1/3 for spherical structures to 0.6 for linear polymers).23m = 1/3, M) equal to 0.45, which corresponds to a 2.8-fold increase in the molecular weight for the unknown assembly compared to complex CB[7]·1, in remarkably accurate agreement with a possible trimerization process.While no aggregation is observed with guest 1 and CB[7] in D26 and commercial sources), and determined their sodium content by inductively coupled plasma optical emission spectrometry (ICP-OES). All solutions returned a sodium content of ca. 0.065% which corresponds to one Na+ per ca. 30–35 CB[7] units. All efforts to remove sodium by dialysis failed to decrease sodium content; whether the latter might in fact originate from glassware etching during the preparation and storage of CB[7] remains an open question. To the best of our knowledge, unlike hydrochloric acid and water contaminations, sodium contamination of CB[n] batches has not been reported before. This is easily understandable as those cations typically do not significantly affect recognition processes in aqueous solution.The presence of small amounts of the possible trimer in the absence of added sodium cations remained perplexing and lead us to suspect a possible contamination of CB[7] sources with sodium cations, a very common occurrence whenever glassware is used. To test this hypothesis, we prepared a series of CB[7] solutions in water at known concentrations  using a sample containing a mixture of complex CB[7]·1 and its unknown parent. Adjusted for their respective concentrations, 5.6- and 3.8-fold increases were observed in the volume integrations of the cross-peaks between the pseudoequatorial methylene hydrogens H3·Na]4+ (m/z 1106.2). The trimeric species were detected also as [(CB[7]·1)2·(CB[7]·Na)·Na]4+ (m/z 1035.9), [(CB[7]·1)3·Na·NaCl]4+ (m/z 1120.2) and [(CB[7]·1)2·(CB[7]·Na)·Na·DMSO-d6]4+ (m/z 1056.4). Other oligomeric species, such as [(CB[7]·1)2·Na]3+ (m/z 985.7), [(CB[7]·1)4·Na]5+ (m/z 1178.4), [(CB[7]·1)5·Na]6+ (m/z 1226.0) and monomer [(CB[7]·1)·Na]2+ (m/z 744.8) were significantly less abundant. We note the absence of coordinating DMSO in the trimer and higher oligomers, as the CB[7] portals fully occupy the first solvation shell of the sodium cation.Finally, sodium cation-promoted trimerization was confirmed by electrospray ionization mass spectrometry . The mosn dissociation analysis of the parent ion [(CB[7]·1)3·Na]4+ also suggested high stability in the gas phase. The fragmentation pathway consists of three subsequent losses of the guest molecules 1, that were sequentially expelled from the CB[7] cavity ([(CB[7]·1)3·Na]4+ → [(CB[7]·1)2·CB[7]·Na]3+ → [(CB[7]·1)·CB[7]2·Na]2+ → [CB[7]3·Na]+). Remarkably, the trimeric cluster remained intact, and each following fragmentation required higher collision energies 12) and sodium 2,3,4,5,6,7,8,9,10,11-undecamethylcarba-closo-dodecaborate (NaHCB11(CH3)11), which have the unique advantage of allowing direct concentration determination of sodium by 1H NMR spectroscopy using the carborane hydrogen signals. Addition of calcium nitrate, calcium triflate and barium nitrate also afforded trimerization – the role of the anion will be discussed later. We also note that the sodium-secured trimer remains intact in the presence of an excess amount of 15-crown-5 (i.e. the latter does not compete with CB[7] for sodium binding). Similarly, adding a 5 : 1 mixture of 15-crown-5 and NaCl to complex CB[7]·1 affords the trimer.Trimerization was observed with the chloride salts of sodium, potassium, calcium, strontium, and europium, but not lithium, rubidium, cesium, magnesium, cerium or ammonium; it was also observed with sodium dodecamethylcarba-β13 ]3 as a function of [M] should return a straight line. This is not the case (see 11(CH3)11)! Straight lines were not obtained either when considering the putative formation of cation-secured dimers (CB[7]·1)2M or simple adducts CB[7]·1·M or a combination thereof. The excellent goodness-of-fit of the quadratic trend in β23 (27–29 in conjunction with the ALPB solvation model30 for DMSO).The concentrations of cation-secured trimer (CB[7]·1)d in β23  in which3M]/[(CB[7]·1)]3 as a function of [M][X]  returns excellent linear correlations (R2 = 0.998 for NaHCB11(CH3)11, Plotting [(CB[7]·1)β23 (in M−4) for all cations that afford the trimeric aggregate are listed in 14 M−4); a 2.4-fold decrease is measured with potassium chloride compared to sodium chloride. Alkali-earth cations afford much lower equilibrium constants (up to 1.9 × 1011 M−4 for strontium), followed by an approximately 80-fold decrease with calcium and further 15-fold decrease with barium. In each series, ionic radii of 1.0–1.3 Å return the highest equilibrium constants, while significantly smaller cations (like lithium and magnesium) or larger cations (like rubidium and cesium) do not allow trimerization.Equilibrium constant We note that the equilibrium constant for the formation of the potassium-secured trimer was obtained using a competition experiment between the sodium-secured trimer and potassium chloride; both aggregates can be readily identified Fig. S73.73·M–X and CB[7]·1 are the preponderant species in solution, extreme cooperativity must be present with equilibrium constant K3 becoming far greater than K2 and K1. Evidence for cooperativity is provided by the 1H NMR titration experiments. If K1, K2 and K3 were similar and significantly above 103 M−1, trimer (CB[7]·1)3M would be the dominant species upon addition of 0.33 equiv. MCl, dimer (CB[7]·1)2M after addition of 0.67 equiv. MCl and finally complex CB[7]·1·M in the presence of at least 1.0 equiv. MCl (as ion pairs or not). This is clearly not the case here: for example, upon addition of 0.33 to 1.0 equiv. NaCl, the ratio of trimer (CB[7]·1)3·M–X vs. complex CB[7]·1 increases steadily from 0.5 to 3.3.To rationalize the formation of the cation-secured trimers, we decompose equilibrium i.e. in water and with the relevant cations in DMSO, cation binding to complex CB[7]·1 can be fitted with a 1 : 1 binding model using equilibrium K1 must be significantly higher than K2 and K3, i.e. negative cooperativity is observed. We do not have evidence for the formation of ion pairs in these cases. As complexes CB[7]·1 and CB[7]·1·M show distinct UV-Vis absorption features in both solvents, a collection of spectra obtained upon addition of aliquots of salts could be fitted with a 1 : 1 binding model to extract equilibrium constant K1, assuming no cooperativity between both CB[7] portals in the absence of guest, and no trimer formation .32 Binding affinities of alkali cations towards CB[7] in water range from log K = 2.3–3.5, and doubly charged alkali-earth cations from log K = 3.2–5.3 (see K(CB[7]·1)/K(CB[7]) consistently lower than 1). We attribute this difference to weak interactions between the cations and the hydrophobic adamantyl unit upon cation binding to the CB[7] rim. To the contrary, in the absence of any guest, the few water molecules present in the CB[7] cavity must provide additional solvation to the cation. Cation binding affinities for CB[7] in DMSO are significantly lower than in water (down to 1900-fold for Ba2+ but the median decrease is approximately 36-fold). However, remarkably, encapsulation of guest 1 enhances the affinity of the assembly towards cations in DMSO methanamine (8.9 × 108 M−1).8A set of 4 additional CB[7] guests were tested for cation-secured trimer formation in DMSO  and 9; m3Na–Cl to assembly (CB[7]·4)3Na–Cl Na–Cl, (labelled “411”), and (CB[7]·1)(CB[7]·4)2Na–Cl (labelled “441”). Three 1H NMR signals for the trimethylsilyl units corresponding to homo-assembly (CB[7]·4)3Na–Cl and the pair of hetero-assemblies can be isolated Finally, we used guest 5 as a negative control, as both CB[7] portals in complex CB[7]·5 interact with the guest's pyridinium groups. Complex CB[7]·5 was indeed insensitive to the addition of NaCl and the cation-secured trimer did not form.2O. To justify this effect, we assessed equilibrium 3Na]4+.A key feature of the trimerization is its solvent-dependency: it is only observed in DMSO and not in Di.e. at 0 K) in the gas phase ΔE, (2) the enthalpic and entropic correction at 25 °C δΔG, and (3) the solvation contributions ΔGsolv  the electronic contributions to the equilibrium −1 at 0 K and by 63.6 kcal mol−1 at 25 °C, that includes the mostly entropic penalty for aggregation, see 39 and Dearden40 who have shown significant aggregation of CB[n]s in the gas phase. The penalty for the partial desolvation of 3 (out of 6) CB[7] portals during the trimerization is overwhelming, however  due to coulombic interactions between the cation and at least 2 carbonyl groups of each CB[7] macrocycle. The desolvation penalty of the sodium cation and of half of all CB[7] portals is of course very penalizing , but still makes the trimerization process slightly favorable in solution . While we do not observe trimerization in water, a 4 kcal mol−1 error on such large favorable or unfavorable contributions is perfectly acceptable; furthermore, calculations correctly predict that the trimerization is more favorable in DMSO! Calculations with potassium afford an unfavorable trimerization in water , and a favorable one in DMSO , in sharp agreement with experimental results , yet the desolvation penalty is just as strong; the overall trimerization is thus unfavorable in either solvent, again in agreement with experiments.In the absence of cation, GFN2-xTB calculations show that trimerization is favorable in the gas phase  in DMSO compared to water by 35.6 kcal mol−1, but three complexes CB[7]·6, with six solvent-exposed portals, are less solvated by 44.4 kcal mol−1, bringing the solvation balance to 8.8 kcal mol−1 in favor of the trimerization in DMSO (3Na is less solvated (i.e. less stabilized) in DMSO compared to water by just 28.2 kcal mol−1, but again, complex three complexes CB[7]·6 are less solvated by 44.4 kcal mol−1, bringing the solvation balance to 16.2 kcal mol−1 in favor of the trimerization in DMSO; however, the desolvation penalty of the sodium cation is more penalizing in DMSO37,38 , bringing back the solvation balance to just 10.5 kcal mol−1, again in favor of the trimerization in DMSO . The DMSO/water balance is even more shifted towards trimerization in DMSO with potassium  in DMSO . This is+ (three sodium cations interacting with three polar solvent molecules) compared to assembly [(CB[7]·G)3·Na]+ (only one sodium cation interacting with three polar solvent molecules). This effect would significantly shift equilibrium Continuum solvation models do not allow a proper determination of solvation energies for macrocycles with deep and small cavities that can only accommodate a few discrete solvent molecules. Therefore we cannot use the methodology proposed above to explain why free CB[7] does not afford trimers in any solvent, regardless of the nature of the cation. Using equilibrium 3 trimer essentially behaves as a cryptand with exceptional affinity for cations of adequate volumes in DMSO. All cations that promote trimerization have cationic radii ranging from 0.98 Å (Na+) to 1.43 Å (Ba2+), while Li+, Mg2+, Rb+, Cs+ and NH4+  are either too small or too large to fit within the trimer central pocket cket see .41,423·Na]+ and the chloride anion using equilibrium + and Cl− anions from the gas phase to DMSO 37 was obtained from the lattice energy of NaCl in the solid state and its solubility in DMSO. The error on the calculated solvation energy of the ion-paired assembly  is likely high, as the anion remains partially exposed to the solvent, and the COSMO-RS formalism is not designed to return accurate solvation energies for charged species directly exposed to the solvent. Calculations return 32 kcal mol−1 for the strength of the ion-pair interaction, which is obviously a vast overestimation; but it certainly does not invalidate its formation.Finally, calculations support the formation of an ion pair between assembly [(CB[7]·6)+, K+), alkali-earth  or at least one cation of the lanthanide series (Eu3+) is added to the solution, and (2) the cavity of CB[7] is filled with a guest that leaves one carbonylated portal available for cation binding. In other terms, the CB[7]/guest trimer, with its carbonyl groups pointing towards its inner core, acts as a cryptand with exceptional affinity to these cations in DMSO. The driving forces for the trimerization are (1) coulombic interactions between the 3 carbonylated CB[7] rims and the cations, and (2) favorable CH–O interactions between the carbonylated rim of a CB[7] macrocycle and the equatorial and pseudoequatorial hydrogens of a neighboring CB[7] unit. In addition to the entropically unfavorable aggregation, free energy penalties are (1) the desolvation of the cations upon nesting into the trimer, and (2) the desolvation of 3 out of 6 CB[7] carbonylated rims upon trimerization. The latter is significantly less unfavorable in DMSO compared to water or deuterium oxide, hence the solvent-selective process. This study paves the way for the design, and subsequent assessment of their recognition properties, of well-defined two- and three-dimensional CB[n]-containing clusters in organic solvents.We have shown that CB[7] forms well-defined trimeric assemblies in DMSO, as long as (1) a selection of alkali- (NaAll analytical data is provided in the narrative and in the ESI.†J. K. and E. M. conceived the project. D. L., F. M., J. Š. and M. D. carried out all experiments. S. G., B. J. B. and J. C. provided mass spectrometry support. I. C. determined all single-crystal X-ray diffraction structures. E. M. carried out all computational work. E. M. and J. K. wrote the manuscript through contributions of all authors.There are no conflicts to declare.SC-014-D3SC02032K-s001SC-014-D3SC02032K-s002


KaH+ values as high as32.2 in acetonitrile, which is up to 13 pKaH+ units greater than the pKaH+ values of the non-interlocked components, and bringsthe free base rotaxanes into the basicity realm of phosphazene superbases.The rotaxane superbases are generally chemically stable and, in amodel reaction for superbases, eliminate HBr from a primary alkylbromide with complete selectivity for deprotonation over alkylation.Their modest size, ease of synthesis, high basicity, low nucleophilicity,and, in the best cases, rapid substrate deprotonation kinetics andexcellent hydrolytic stability make compact amine-crown ether rotaxanesuperbases intriguing candidates for potential applications in synthesisand supramolecular and materials chemistry.Challenges for the development of efficacious new superbasesincludetheir ease of synthesis, chemical stability, and high basicity, whileminimizing nucleophilicity is important for reducing unwanted sidereactions. Here, we introduce a new family of organic superbases,compact amine-crown ether rotaxanes, which show desirable characteristicsin all these respects. Metal-free active template synthesis providesaccess to a range of rotaxanes with as little as three atoms betweenthe stoppering groups, locking the location of a small crown ether(21C7 and 24C8 derivatives) over the amine group of the axle. Theforced proximity of the interlocked protophilic components resultsin p In the amine form, the crown etherusually has sufficient positional flexibility along the axle thatit can adopt co-conformations that avoid strong electrostatic repulsionbetween the nitrogen and oxygen lone pairs.25 However, the recently developed metal-free active template synthesis30 enables the facile synthesis, under kinetic control, of compactrotaxanes31 in which the location of thering is locked in place on a very short axle, forcing the protophilicelements of the ring and thread into close proximity. Here, we reporton a new family of organic superbases: compact amine-crown ether rotaxanes.In these structures, co-conformational freedom of the components isrestricted, and a destabilizing effect arises from electrostatic repulsionbetween the proximate nitrogen and oxygen lone pairs, providing agreatly enhanced thermodynamic driving force for protonation (Hydrogen bonding between ammonium groups andcrown ethers is acommon way to template (pseudo)rotaxane assembly 1A.15−19 tonation 1B. Addit24C8⊂3, which was prepared ina single step as its HBr salt using metal-freeactive-template synthesis .We began our investigationswith the rotaxane ynthesis 1.26 Libe1H NMR spectrum of rotaxane 24C8⊂3·HPF6 in CD3CN featuressignals at 4.76(Hc), 8.11 (Hb), and 8.20 (Ha) ppm signal moves substantially upfield to 4.31 ppm . In comparison, in the free thread 1 , the benzylic methylene and aromatic proton signals areat 3.90, 7.84, and 7.90 ppm, the crown ether significantly deshieldingthe internal region of the short axle (Ha/a′ andHc/c′) in both the protonated and deprotonated formsof the compact amine-crown ether rotaxane.The (Ha) ppm2. Upon d8 thepKaH+ of 24C8⊂3 was determined as 20.5 ± 0.1 in CD3CN, using 1,4-diaminobutane(pKaH+ = 20.1 in CD3CN)1 as a reference base . In contrast,the secondary amine in the free thread (3) has a pKaH+ of 12.0 ± 0.3, showing thatthe crown ether has a very substantial influence on the pKaH+ of the compact rotaxane. The pKaH+ value of 20.5 ± 0.1 in CD3CN of rotaxane 24C8⊂3 is at the lower limit oforganic superbases.Using Lambert’s method,We next prepared a series of rotaxanes withvarious tight macrocyclesand different stoppers to see if we could further enhance the amine-crownether rotaxane basicity. Each rotaxane was synthesized through metal-freeactive template synthesis 1. The prKaH+ decreases marginally to 20.3± 0.1 whenaxle 3 is enclosed by a dibenzo-24-crown-8 macrocycle(rotaxane DB24C8⊂3), despite 24-crown-8 bindingdibenzylammonium threads substantially stronger than their dibenzo-24-crown-8homologues.32 The enthalpic effect of dibenzylammoniumbinding appears to be less important for the resulting pKaH+ than the increased preorganization andrigidity of the dibenzo-crown and the consequences that that has onlone pair repulsion between the thread and the macrocycle. The pKaH+ increases dramatically to 24.9± 0.1 when a smaller macrocycle 21-crown-7 (rotaxane 21C7⊂3) is used  resulted in a pKaH+ increase from 20.5 ± 0.1 to 22.9 ± 0.1 (p-methoxy unit to one of the stoppers (rotaxane 24C8⊂5) gave a marginal increase in pKaH+ to 23.2 ± 0.2, but at the expense of a lower rotaxaneyield  resulted in a significant pKaH+ enhancement to 26.3 ± 0.2, althoughthe isolated rotaxane yield was reduced . The pKaH+ of rotaxane DB24C8⊂8 was measured to be 20.7 ± 0.3 in CD3CN. This is slightly higher than that of DB24C8⊂3, with the decrease in pKaH+ due to the second binding site and slightly longer axle apparentlycompensated for by the replacement of a 3,5-bis(trifluoromethyl)phenylstopper with a more electron-rich alkyl group. Rotaxane 21C7⊂6, featuring the tightest macrocycle in the series with electron-richstoppers, was synthesized in one step in a modest 10% yield. The pKaH+ of 21C7⊂6 was measured to be 32.2 ± 0.6 in CD3CN, more basicthan many phosphazene superbases2 and similarto Verkade superbases.9Rotaxane DB24C8⊂8 3 was syn2Cl2, a less competitive solvent than CD3CN with regard to hydrogen bonding . We found that the pKaH+ values of the compact rotaxane superbasesappeared to be increased in the less polar solvent relative to conventionalsuperbases.We also measuredthe basicity of an amine-crown ether rotaxanein CD24C8⊂3·HBr, DB24C8⊂3·HPF6, and 21C7⊂3·HBr (S3–S5). The solid-state structure of 24C8⊂1·HBr from one of the −N+CH2– groups to the crown ether. The DB24C8 macrocycle is folded to allow π-stacking betweenan electron-rich catechol of the crown ether and one of the electron-deficient3,5-bis(trifluoromethyl)aryl stopper groups of the axle . Each features intercomponent hydrogenbonds similar to that of 24C8⊂3·HBr withhydrogen bonds formed betweenthe N–Hs and several glycolic oxygens phosphorane(P1-tBu)  fast kineticsof deprotonation of other species, (ii) low nucleophilicity, and (iii)good hydrolytic stability. We investigated these properties in someillustrative experiments that compared the rotaxane efficacy in theserespects to that of some common conventional organic superbases, 1,8-diazabicyclo(5.4.0)undec-7-ene(DBU), 7-methyl-1,5,7-triazabicyclo(4.4.0)dec-5-ene, and phosphazenebase (P1-tBu) 1 and 2.9a was chosen to exemplify relativerates of deprotonation by the superbases and their tendency for promotingelimination over nucleophilic addition as an unwanted side reaction(S1). A primary alkyl bromide was used as thesubstrate so that the competing SN2 nucleophilic additionto form 9b provided a reasonable kinetic and thermodynamicalternative to E2 elimination.37 None of the rotaxane superbases,and only DBU of the conventional superbases, yielded any 9b in this experiment are ongoing.Two of the rotaxanes, at 25 °C 1. Even as faster 1. Althou3OD/D2O with2 mol % pivalic acid as an internal standard (S2). Fourof the trialed rotaxanes showed no evidence of any degradation underthese conditions, even at 80 °C for more than a week. Rotaxane 24C8⊂2 significantly decomposed over a few hours atroom temperature (t1/2 = 6 h), and 21C7⊂3 was consumed completely within a matter of minutes macrocycle, is stable, as is axle 3 when encapsulated by other macrocycles . What makescomponents 21C7 and 3 particularly reactivein hydrolytic media when threaded is unclear. We note that the tworotaxanes with very slow kinetics for deprotonation of 4-bromophenethylbromide are also the two rotaxanes that are hydrolytically unstable.Apart from these two examples, the hydrolytic stability of the rotaxanesuperbases compares favorably to DBU and mTBD and is similar to P1-tBu  and the functionality of the dibenzylaminestoppers (electron-rich substituents tend to result in more basicrotaxanes). Two of the compact rotaxanes studied, 24C8⊂2 and 21C7⊂3, deprotonate substrates much moreslowly than the other rotaxanes in the series. These two rotaxanesalso decompose quickly in hydrolytic media, while all the other rotaxanesuperbases are chemically stable. The reasons for the difference inchemistry between 24C8⊂2 and 21C7⊂3 and the other rotaxane superbases studied remain unclear. However,the most basic rotaxane measured, 21C7⊂6, showsexcellent chemical stability under such conditions and has a pKaH+ of 32.2 ± 0.6 in CD3CN, a basicity ∼13 orders of magnitude higher thaneither of its non-interlocked components. The modest size, ease ofsynthesis, high basicity, low nucleophilicity, and, in the best cases,rapid deprotonation kinetics and excellent hydrolytic stability ofcompact amine-crown ether rotaxanes are attractive characteristicsfor potential applications in organic synthesis and other fields ofchemistry. They add to the growing list of remarkable property effectsthat can be introduced through the nature of the mechanical bond.15In summary, we have synthesized and carriedout preliminary studiesof the properties of a new family of organic superbases, compact amine-crownether rotaxanes with as little as three atoms between the axle stoppers.The rotaxane superbases are prepared by kinetically controlled metal-freeactive-template synthesis, generally in one step from commerciallyavailable building blocks, in 10–55% yield. Their p


Inversely, if the crystal lattice volume was modulated post-synthetically, the spin state of the compound could be tunable, resulting in the inverse effect for SCO. Herein, we demonstrate such a spin-state tuning in a breathing cyanido-bridged porous coordination polymer (PCP), where the volume change resulting from guest-induced gate-opening and -closing directly affects its spin state. We report the synthesis of a three-dimensional coordination framework {[FeII(4-CNpy)4]2[WIV(CN)8]·4H2O}n , which demonstrates a SCO phenomenon characterized by strong elastic frustration. This leads to a 48 K wide hysteresis loop above 140 K, but below this temperature results in a very gradual and incomplete SCO transition. 1·4H2O was activated under mild conditions, producing the nonporous {[FeII(4-CNpy)4]2[WIV(CN)8]}n (1) via a single-crystal-to-single-crystal process involving a 7.3% volume decrease, which shows complete and nonhysteretic SCO at T1/2 = 93 K. The low-temperature photoswitching behavior in 1 and 1·4H2O manifested the characteristic elasticity of the frameworks; 1 can be quantitatively converted into a metastable HS state after 638 nm light irradiation, while the photoactivation of 1·4H2O is only partial. Furthermore, nonporous 1 adsorbed CO2 molecules in a gated process, leading to {[FeII(4-CNpy)4]2[WIV(CN)8]·4CO2}n (1·4CO2), which resulted in a 15% volume increase and stabilization of the HS state in the whole temperature range down to 2 K. The demonstrated post-synthetic guest-exchange employing common gases is an efficient approach for tuning the spin state in breathing SCO-PCPs.Iron( Variation of three different stimuli: the type of guest molecules, temperature and light enables intentional manipulation of the mechanical, electronic and magnetic properties of a cyanido-bridged coordination polymer. The prospective candidate can be found in the family of octacyanidometallate-based compounds {[FeII(py)4]2[MIV(CN)8]·xH2O}n ,23 since some of us recently demonstrated large water-induced breathing of the octacyanomolybdate(iv) coordination polymer.24 The impact of guest molecules on SCO properties of octacyanidometallate frameworks was mostly overlooked, with the exception of the water-induced spin transition reported by Song et al. in {[FeII((3-pyridyl)methanol)4]2[WIV(CN)8]·4H2O}n.25 Herein, we report {[FeII(4-CNpy)4]2[WIV(CN)8]}n , a flexible nonporous framework characterized by a weakly cooperative spin transition at T1/2 = 93 K. Upon adsorption of water molecules, it showed a gate-opening breathing transition with a 7.3% increase of crystal volume resulting in {[FeII(4-CNpy)4]2[WIV(CN)8]·4H2O}n (1·4H2O). The reorganization of the crystal framework led to the appearance of a 48 K wide hysteresis loop in the 187–235 K range, accompanied by strong elastic frustration, with the low-temperature phase showing a mixed low-spin/high-spin state. The most striking feature of the nonporous 1 is the ability to accommodate four CO2 molecules per formula unit in a gate-opening fashion, leading to {[FeII(4-CNpy)4]2[WIV(CN)8]·4CO2}n (1·4CO2). Finally, this CO2 uptake resulted in a 15% increase in crystal volume, which in turn stabilized the HS state in the whole temperature range of 2–300 K. Herein, we demonstrate the total control of the spin state of a PCP driven by guest-dependent breathing-induced steric effects.The spin cross-over (SCO) phenomenon is a transition between low-spin (LS) and high-spin (HS) states of metal ions induced by a physical stimulus such as temperature, pressure, or light irradiation.l volume , as wellre phase . We expeII(4-CNpy)4]2[WIV(CN)8]·4H2O}n (1·4H2O) was obtained as orange crystals by a wet chemistry approach . It crystalized as a 3-D coordination network in a tetragonal crystal system, space group I41/a (Table S1ii) centres in the structure of 1·4H2O are surrounded by six nitrogen atoms and equivalent by symmetry. The iron(ii) coordination sphere consists of four nitrogen atoms of aromatic 4-cyanopyridine rings and two cyanido ligands in a trans configuration  in a high-spin (HS) state.25–34 Octacyanidotungstate(iv) forms four cyanide bridges with iron(ii), leaving four terminal cyanides engaged in hydrogen bonding with water molecules with a CN⋯O distance of 2.867(5) Å (Table S235 while the kinetic diameter of water is estimated to be 2.65 Å).36 Noteworthily, the water molecules also form O⋯NC contact with the nitrogen atom belonging to the nitrile group of 4-cyanopyridine (3.074(7) Å). Despite the aromatic nature of the pyridine rings, no π–π interactions are observed, since all pyridine molecules are separated by more than 4 Å. However, 4-cyanopyridine molecules interact with each other through strongly polarized nitrile groups, which form –CN⋯C contacts characterized by the 3.244(5) Å intermolecular distance. Cooling the crystal of 1·4H2O below 190 K led to an immediate increase in crystal mosaicity and the disappearance of high-resolution (<1.0 Å) diffraction peaks and therefore the crystal structure could not be analysed at lower temperatures.The hydrated compound {2[WIV(CN)8]}n (1) was cooled and the sc-XRD measurement was performed at 200 K. Upon dehydration, the symmetry changes from I41/a in 1·4H2O to I4̄2d in 1 and the unit cell volume is reduced by 7.3%. In the dehydrated framework, there are four inequivalent nitrogen atoms coordinated to the Fe(ii) centre  at 200 K, similarly to the framework before dehydration. Importantly, as a result of water removal, the cyanido bridges underwent distinct bending, with the N–C–Fe angle changing from 170.8(2)° to 162.4(7)°. On the other hand, the equatorial pyridine coordination became more ordered, as Npyridine1–Fe–Npyridine2 angles change from 87.73(9)° and 92.27(9)° in 1·4H2O to 90.0(2)° in 1. The aforementioned changes lead to the reorganization of the coordination skeleton as visible along the c-axis, which is depicted in a axis  and gate-closing (GC) processes were recently demonstrated in amorphous38 and microcrystalline24,39,40 cyanido-bridged frameworks, to our knowledge the transition from 1·4H2O to 1 constitutes the first example of gate-closing visualized by single-crystal XRD in a coordination polymer featuring only cyanido bridges.Following the TGA results, the single crystal of the hydrated framework 1·4H) centre , compares Fig. S3. The sel2O transformation is further confirmed by the measurement of volumetric adsorption of water vapor for 1 at 288–308 K 4]2[WIV(CN)8]·4H2O}n formulation deduced for 1·4H2O from sc-XRD measurement. A small further increase in adsorption occurs at higher vapor pressures, which probably results from water adsorption on the surface of crystallites. In the desorption branch, the adsorbed amount of water slowly decreases until it reaches 4.25 mol mol−1 around 35% relative humidity. Below this pressure, an abrupt decrease in the amount of adsorbed vapor corresponding to the GC process can be observed.The gate-opening/closing character of the 1 ↔ 1·4H88–308 K . Adsorpt−1 range and the H–O–H bending band at 1629 cm−1, both confirming the presence of water molecules engaged in hydrogen bonding with cyanide ligands in the structure of 1·4H2O. Furthermore, the nitrile stretching bands of 4-cyanopyridine molecules change their structure  spectrum of 1 Fig. S4a. Water a Fig. S4b, with tw2O and 1 being composed of paramagnetic Fe(ii) ions (S = 2). Consequently, 1·4H2O is characterized by magnetic susceptibility and temperature product (χT) of 7.16(11) cm3 K mol−1 at 265 K (ii) centres per Fe2W formula unit and the Landé factor gFe = 2.18(2). A relatively fast decrease of χT was observed below 192 K, reaching 5.23 cm3 K mol−1 at 140 K. Below this point, a decrease of χT was very gradual, reaching 4.19 cm3 K mol−1 at 60 K . All changes of χT product in the 192–60 K range are attributed to the spin cross-over phenomenon, which is confirmed by Mössbauer spectroscopy measurements performed on cooling /dT derivative) equal to 187 K on cooling and 235 K on heating , the resulting transformation enthalpy is ΔHSCO = 8.5–11.4 kJ mol−1 Fe, leading to ΔSSCO = 38–51 J K−1 mol−1.Structural data at 200 K are consistent with both 1·4Hat 265 K , which c heating , inset.  heating  and dyna heating . The sim2O, room temperature χT product observed for 1 equals 7.56(18) cm3 K mol−1 (ii) with gFe = 2.24(3) cm3 K mol−1. No change in χT is observed down to ca. 150 K, below which point the gradual decrease starts. Below 100 K a fast decrease occurs reaching 0.38 cm3 K mol−1 at 50 K. The observed change represents 95% of the room temperature signal and is interpreted as a complete SCO transition, in line with the results of Mössbauer spectroscopy  kJ mol−1, ΔSHL = 51.0(3) J mol−1 K−1 and Γ = 1.12(1) kJ mol−1 with R2 = 0.99999. The determined SCO temperature TSCO = ΔHHL/ΔSHL = 93(1) K is very low, which for the ΔSHL in the typical range (40–80 J K−1 mol−1)44 must result from small ΔHHL. Typically, SCO compounds that demonstrate a spin transition below 100 K become kinetically trapped in the HS state due to the slow dynamics of the spin transition.45–48 However, 1 demonstrates exactly the same SCO behaviour also for a higher temperature sweep rate of 2 K min−1 4(μ-NC)2] moieties.25–34 As the observed contraction of bond lengths falls in the typical range for similar assemblies, this raises the question about the source of unusually small ΔHHL. This could be explained by stabilization of the HS state or destabilization of the LS state. No such effect can be easily identified by comparison of intermolecular interactions in the structures of 1 at 140 and 80 K  centres that undergo the SCO phenomenon. This suggests relatively slow kinetics of hysteretic SCO for 1·4H2O around 150 K, compared to the fast and gradual SCO observed for 1 below 100 K, leading us to two conclusions. First, given the slightly cooperative nature of SCO in 1, which comprises only [–NC–WIV(CN)6–CN–] bridges as elastic interaction pathways, elastic frustration of 1·4H2O must arise from the additional source of antiferroelastic interactions.50 Most likely the appearance of hydrogen bonds increases cooperativity of the spin transition, but water molecules themselves act as stiff rods separating iron(ii) centres. Taking into account the lack of free void spaces in the nonporous 1, which accommodates water vapor only in the pore-opening transition, the steric effect of H2O molecules must prevent the contraction of the framework to the full LS state. As a result, SCO for 1·4H2O is incomplete  centres) at low temperatures. On the other side, the unusually fast kinetics of the SCO transition observed for 1 below 100 K suggests a high degree of structural flexibility, which is confirmed by observation of the pore-opening behaviour induced by water vapor.Although both 1·4Hp Fig. S9, yet it 2 and CO at 77 K, as well as NO at 121 K , which corresponds to approximately 4 CO2 molecules per Fe2W formula unit. This amount is also reflected in the isobar measurements of CO2 adsorption depicted in 2-adsorbed phase from adsorption experiments is {[FeII(4-CNpy)4]2[WIV(CN)8]·4CO2}n (1·4CO2), very similar to the composition observed for 1·4H2O from sc-XRD. Powder X-ray diffraction measurements performed in situ under 100 kPa CO2 on cooling show the disappearance of the parent phase 1 and the appearance of a new phase below 215 K , ΔHGO = −25.0(8) kJ mol−1 and AGC = 16.8(4), ΔHGC = −23.0(7) kJ mol−1. These parameters were used to prepare the phase diagram of the 1/CO2/1·4CO2 system presented in This approach results in 2 resembles the powder pattern of 1·4H2O, although with two apparent differences  Å, b = 19.950(3) Å, c = 16.461(3) Å with Rwp = 1.83%, Fig. S15a/a = 6.1%, Δb/b = 3.9% and Δc/c = 4.1%). The total 14.8% volume expansion from 1 (V200K = 5827.2(3) Å3) to 1·4CO2 (V200K = 6687(3) Å3), corresponds to 860 Å3. For Z = 4 (as observed in 1 and 1·4H2O), this is in line with {[FeII(4-CNpy)4]2[WIV(CN)8]·4CO2}n formulation, as 16 CO2 molecules per formula unit are expected to occupy ≈744 Å3.The powder X-ray diffraction pattern observed for 1·4CO Fig. S14. All ref2, we performed in situ IR and Raman spectroscopy studies under a CO2 atmosphere. Under in situ IR measurement conditions the octacyanidotungstate(iv) cyanide stretching mode is well visible  after gate-opening is hard to explain on its own, while the very strong bands of gaseous CO2 obscure the nitrile stretching bands of 4-cyanopyridine molecules. This issue is resolved by Raman spectroscopy measurement, in which no signal of gaseous CO2 is observed in the 2300–2000 cm−1 range . A sample of 1·4H2O was activated inside the magnetometer chamber and the gas cell was filled with 100 kPa He to facilitate good thermal contact. Then magnetic susceptibility was studied in the 250-5 K range , while the CO2 resublimation that starts below 188 K results in a large continuous drop of pressure. The observed temperature range of adsorption in the magnetic measurement is downshifted by ≈10 K as compared to the isobar adsorption measurement under 100 kPa CO2  centers and the zero-field splitting effect. In order to test the reversibility of the magnetic switching behavior, the sample was activated again at 323 K and the experiment was repeated under 50 kPa CO2  and M(H) curves can be satisfactorily described with the following Hamiltonian constructed in the PHI software52  centers are in the high-spin state: g = 2.27(5), D = 9.0(1) cm−1, E = 2.3(1) cm−1, with the additional intermolecular interaction parameter zJ = −0.085(1) cm−1. The obtained values are within the reasonable range for a HS iron(ii),53–56 which clearly confirms that the magnetic behavior of 1·4CO2 can be properly described without assuming any degree of spin cross-over.The experimental curves are well reproduced by the following set of parameters, assuming all iron(ii) spin cross-over compounds, and as far as we know was never observed with CO2 as guest molecules. Although CO2 adsorption in SCO compounds was previously reported in the literature, it was found to have negligible impact on the magnetic properties of Hofmann-type frameworks10,11 or lead to a small modulation of spin transition temperature in other compounds.57,58 The only exception was observed recently by Hayami et al. in a monomeric CoII compound, where CO2 adsorption stabilizes the LS state by strong intermolecular interactions.59 On the other hand, the complete stabilization of the HS state in 1·4CO2 is expected to originate from the large volume expansion triggered by CO2-induced gate-opening  sites, with CO2 molecules acting as long rigid rods, preventing network contraction associated with the spin transition. As such, this effect should not be restricted to CO2 only, but would also be expected for other gas molecules adsorbed in the breathing frameworks. Although we did not observe breathing behavior for 1 under a N2, CO or NO atmosphere  effect.60 To allow for efficient irradiation and preserve the anhydrous state in the magnetometer chamber, the polycrystalline sample of 1 was crushed in an agate mortar and sealed inside a polyethylene bag . The as-prepared sample reaches a slightly higher value of 1.15 cm3 K mol−1 at 30 K, compared to 0.38 cm3 K mol−1 in bulk ,61,62 as demonstrated in χT product reaches a maximum of 7.02 cm3 K mol−1 at 35 K, which is very close to the 7.56 cm3 K mol−1 observed at room temperature and therefore the LIESST for 1 is concluded to be almost quantitative. Furthermore, the TLIESST determined for 1 as an extremum of d(χT)/dT equals 53 K  centers surrounded by six independent ligands. Both of these characteristics – the completeness of the photo-induced transition to the HS state and the low T0 value – further confirm the high degree of structural flexibility in 1. Moreover, the photoinduced HS state in 1 at 10 K is rather persistent, as in the repeated experiment in which 638 nm was followed by 808 nm irradiation . All the temperature-, light- and sorption-induced changes in the spin state of 1 are summarized in Fig. S27.†Phase 1·4H Fig. S24. The χT  Fig. S26. This maII(4-CNpy)4]2[WIV(CN)8]}n (1). It shows an unusually low temperature of spin crossover transition T1/2 = 93 K, resulting from a very small enthalpy change. Despite the low temperature of the structural transformation, the spin transition is complete and proceeds relatively fast (as compared to other compounds demonstrating low-temperature SCO). This results from flexibility of the framework, which is confirmed by the observation of a gate-opening behavior induced by H2O and CO2 molecules. To the best of our knowledge, the transition between 1·4H2O and 1 is the first example of the gate-closing process studied by single-crystal XRD for a framework with only cyanides playing the role of a bridging ligand (without organic linkers). The breathing behavior resulting from water adsorption leads to the appearance of a 48 K wide hysteresis loop in the case of 1·4H2O, unusual among octacyanidometallate-based SCO compounds. More importantly, the inclusion of CO2, which is assisted by an almost 15% volume expansion upon the transition from 1 to 1·4CO2, results in the stabilization of the high-spin state in the entire temperature range studied. This shows that the “internal pressure” effect of rigid gas molecules can induce spin change in a spin cross-over framework, even in the absence of strong intermolecular interactions.In search of three-dimensional breathing spin cross-over frameworks, we have prepared a coordination polymer {[Feiv) dihydrate was obtained according to the reported procedure.65 All other reagents were supplied by Sigma-Aldrich. Gases used for adsorption measurements were supplied by Nippon Sanso Holdings Corporation .Potassium octacyanotungstate(4[WIV(CN)8]·2H2O  was dissolved in 8 mL H2O, and in the second vial 4-cyanopyridine , Mohr's salt  and ascorbic acid  were dissolved in 24 mL H2O with 15 minutes of sonication in the ultrasonic bath. Then, both solutions were heated for 30 minutes at 36 °C in a water bath. Afterwards, a solution of octacyanotungstate(iv) was dropwise added to the iron(ii) containing mixture with hand stirring. The resulting clear yellow mixture was left at 36 °C to crystalize. The red octahedral crystals that appeared after 48 hours were collected by filtration. Yield: 10 mg (30%). Purity was confirmed by elemental analysis and powder X-ray diffraction : C: 47.75%, N: 23.86%, H: 2.86%. Found: C: 48.72%, N: 24.38%, H: 2.57%. The discrepancy results from partial sample dehydration before the start of the measurement. Calcd for C56H37Fe2N24O2.5W: C: 48.68%, C: 24.33%, H: 2.70%.In the first vial, Kn Fig. S2. Anal. c2O and 1 were performed using a Bruker D8 Quest Eco diffractometer . Single crystals of 1·4H2O were moved directly from the mother liquor into NVH oil to avoid loss of the crystallization solvent. A single crystal of 1 was prepared in situ by heating 1·4H2O mounted on the goniometer head in a dry nitrogen stream using a Cryostream device (Oxford Cryosystems). Absorption corrections, data reduction and unit cell refinements were performed using SADABS and SAINT programs included in the Apex3 suite. The structures were solved using intrinsic phasing and refined anisotropically using weighted full-matrix least-squares on F2.66–68 Hydrogen atoms of the ligands were placed in calculated positions and refined as riding on the parent atoms. Structural diagrams were prepared using Mercury CSD 2020.3.0.35 CCDC 2240344 (1·4H2O at 200 K) and 2240345–2240347  contain the supplementary crystallographic data for this paper.Sc-XRD experiments for 1·4H2O loaded into glass capillaries under mother liquor (0.7 mm in diameter). Sample of 1 was obtained by drying 1·4H2O in a vacuum (p ≈ 10−2 mbar) for 2 hours and inserted into a glass capillary in oxygen- and water-free atmosphere of a glovebox  and sealed with silicone grease. The results were subjected to background correction using the DIFFRAC algorithm implemented in the DIFFRAC.EVA V5 software. PXRD data for in situ CO2 adsorption measurement were collected for samples in 0.5 mm diameter glass capillaries using a Rigaku Ultima IV diffractometer (Cu Kα radiation). In order to allow vacuum treatment and CO2 introduction, the capillary was connected to stainless-steel (SUS) lines connected to a gas-handling system . The temperature was controlled by a N2 gas stream. The results were background corrected by subtracting the pattern recorded for the empty sample stage at room temperature. The unit cell for 1·4CO2 was determined for the uncorrected measurement using FOX software,69 and Le Bail refinement was performed using JANA2006 software.70PXRD data for phase purity confirmation Fig. S2 were obtca. 25 mg sample of 1·4H2O activated by heating to 323 K under vacuum (p ≈ 10−4 mbar). For isotherm measurements of N2 and CO at 77 K and CO2 at 195 K, liquid nitrogen or dry ice/methanol bath was used. In the case of water vapor isotherm temperature was controlled using a water bath. For isobar measurements and isotherm measurements at different temperatures (including NO at 121 K), a home-built cryostat (ULVAC-Cryo) was utilized.The adsorption isotherm measurements were recorded on an automatic volumetric adsorption apparatus  for 2O were recorded using a Nicolet iN10 MX FT-IR microscope in transmission mode . Sample of 1 was obtained by in situ dehydration of 1·4H2O with dry nitrogen purge inside a Linkam THMS350V temperature-controlled stage. In situ CO2 adsorption was studied using a JASCO FT/IR-4200 spectrometer for the sample of 1 mixed with KBr and dispersed in between two CaF2 windows inside the cryostat  connected to a gas-handling and pressure monitoring system . Raman spectra during in situ CO2 adsorption were obtained using a JASCO NRS-4500 Raman microscope using a cryostat system  connected with a gas-handling and pressure-monitoring system . Quartz and CaF2 were equipped over the sample room and the vacuum-insulating shield of the cryostat, respectively. The sample was inspected using an objective lens (Olympus SLMPLN20x) and was irradiated with a 532 nm Raman excitation laser. The transmission 57Fe Mössbauer spectra were collected using a Wissel spectrometer with a liquid nitrogen bath cryostat. A polycrystalline sample of 1·4H2O (ca. 50 mg) was inserted into a polyethylene (PE) bag with mother liquor to prevent its dehydration and sealed using an impulse heat sealer. Sample of 1 (ca. 50 mg) was obtained by drying 1·4H2O in a vacuum (p ≈ 10−2 mbar) for 2 hours, and then sealed inside a PE bag in an oxygen- and water-free atmosphere of a glovebox . The samples were mounted for the measurements inside a copper ring. Mössbauer spectra were fitted with the use of the WinNormos-for-Igor software package, assuming the Lorentzian shape of the resonance lines. In the case of 1·4H2O, two additional doublets  originating from the iron species in solution were included to improve the quality of the fits. Those signals  forms in the solid, which were determined from the ratio of the areas of the corresponding doublets attributed to 1·4H2O.Infrared spectra for 1·4H2O were performed using a Quantum Design MPMS-3 Evercool magnetometer in magnetic fields up to 7 T. Polycrystalline sample of 1·4H2O (10.9 mg) was inserted into a polyethylene (PE) bag with a minimal amount of mother liquor and sealed using an impulse heat sealer. Sample of 1 (8.6 mg) was obtained by drying 1·4H2O in a vacuum (p ≈ 10−2 mbar) for 2 hours, and then sealed inside a PE bag in oxygen- and water-free atmosphere of a glovebox . Both samples were mounted onto the quartz sample holder using Kapton tape. The experimental data were corrected for the diamagnetism of the sample. Samples were prepared for photomagnetic measurements in the PE bags in a similar manner, but only a small amount (ca. 1 mg) was used, and PE bags were placed between two layers of adhesive tape (5 mm diameter) and inserted into the plastic straw. Irradiation was performed using laser diodes (power at the sample position 10–20 mW cm−2). Diamagnetic corrections were determined by comparison with bulk measurements.Magnetic and photomagnetic studies for 1 and 1·4H2 atmosphere were performed using a Quantum Design MPMS-XL on a polycrystalline sample of 1·4H2O (14.9 mg), which was placed inside a gelatin capsule with ca. 100 mg of polyester wool to ensure its immobilization. The capsule was pierced with a needle and placed inside a straw, which was attached to the home-built SUS (stainless steel) sample rod described elsewhere.18 The SUS tube was connected to the gas-handling system . The sample was activated in situ by heating it to 323 K and vacuum pumping (p ≈ 10−4 mbar). Measurements for the sample in the activated state (1) were performed under 100 kPa He to ensure good thermal contact of the sample with the magnetometer cavity. Measurements for 1 under a CO2 atmosphere were performed with an additional 5 kPa He in the system, in order to preserve thermal conductivity after complete CO2 resublimation. The connection of the SUS sample rod to the gas-handling system was left open for the entire measurement time in order to enable constant monitoring of the gas pressure in the system. All the measurements were conducted in the temperature stabilization mode. Diamagnetic corrections were determined by comparison of the measurement for activated 1 with the bulk measurement for 1 in the PE bag.Magnetic susceptibility measurements under a CO−1) and a temperature scanning rate of 2 °C min−1. DSC was performed using a NETZSCH DSC214 Polyma for the sample in a closed-lid sample holder at a temperature sweep rate of 5 K min−1. Elemental analyses were performed using an ELEMENTAR Vario Micro Cube CHNS analyzer.TGA was performed using a NETZSCH TG 209 F1 Libra under a flow of nitrogen , supervision, writing – review & editing. H. Miyasaka: funding acquisition, methodology (in situ CO2 adsorption studies), project administration, writing – review & editing. D. Pinkowicz: conceptualization, funding acquisition, investigation and formal analysis, project administration, writing – review & editing.M. Magott: conceptualization, funding acquisition, investigation and formal analysis , supervision, visualization, writing – original draft, writing – review & editing. K. Płonka: investigation and formal analysis , writing – original draft. B. Sieklucka: funding acquisition, project administration, writing – review & editing. K. Dziedzic-Kocurek: investigation and formal analysis (Mössbauer spectroscopy). W. Kosaka: funding acquisition, investigation (gas adsorption and magnetic measurements), methodology (There are no conflicts to declare.SC-014-D3SC03255H-s001SC-014-D3SC03255H-s002


In mitochondria, protein and ATP synthesis and various metabolic pathways are directly regulated by Mg2+. MRS2, a magnesium channel located in the inner mitochondrial membrane, mediates the influx of Mg2+ into the mitochondrial matrix and regulates Mg2+ homeostasis. Knockdown of MRS2 in human cells leads to reduced uptake of Mg2+ into mitochondria and disruption of the mitochondrial metabolism. Despite the importance of MRS2, the Mg2+ translocation and regulation mechanisms of MRS2 are still unclear. Here, using cryo-EM we report the structures of human MRS2 in the presence and absence of Mg2+ at 2.8 Å and 3.3 Å, respectively. From the homo-pentameric structures, we identify R332 and M336 as major gating residues, which are then tested using mutagenesis and two cellular divalent ion uptake assays. A network of hydrogen bonds is found connecting the gating residue R332 to the soluble domain, potentially regulating the gate. Two Mg2+-binding sites are identified in the MRS2 soluble domain, distinct from the two sites previously reported in CorA, a homolog of MRS2 in prokaryotes. Altogether, this study provides the molecular basis for understanding the Mg2+ translocation and regulatory mechanisms of MRS2.Magnesium ions (Mg 2+ channel MRS2 and identified key residues in gating. This work provides insights into the gating and regulatory mechanisms of MRS2.Magnesium homeostasis is key for cellular functions. Here, authors report structures of human mitochondrial Mg It acts as a cofactor of more than 600 enzymes, including protein kinases, ATPases, exonucleases, and other nucleotide-related enzymes. It has been shown that Mg2+ is involved in various physiological functions such as muscle contraction, vasodilation, neuronal signaling, and immunity3. Intracellular Mg2+ concentrations are tightly regulated and dysregulation of Mg2+ homeostasis is associated with diseases including muscular dysfunction, bone wasting, immunodeficiency, cardiac syndromes, neuronal disorders, obesity, Parkinson’s disease, and cancer6.Magnesium , oxidative metabolism and directly participates in mitochondrial metabolism in form of MgATP2-9. Although the high inner mitochondrial membrane potential (about −180 mV) can theoretically drive a large electrophoretic Mg2+ influx into the mitochondrial matrix, the free Mg2+ concentration in the matrix is found to be comparable to that in the cytosol (~0.8 mM), implying Mg2+ transport is tightly regulated in order to maintain normal mitochondrial physiology10. Most of the Mg2+ influx into mitochondria is mediated by the Mitochondrial RNA Splicing 2 (MRS2) channel, located in the inner mitochondrial membrane14. Knockdown of MRS2 in human cells leads to reduced uptake of Mg2+ into mitochondria, loss of respiratory complex I, disruption of mitochondrial metabolism, and cell death16. A loss of function mutation disrupting MRS2 is also associated with demyelination syndrome in rats17. Recent studies in mice show that MRS2 is required for lactate-mediated Mg2+-uptake in mitochondria18, and knockout of MRS2 causes reprogramming of the metabolism including upregulation of thermogenesis, oxidative phosphorylation and fatty acid catabolism via HIF1α transcriptional regulation19. Despite the physiological implications of MRS2, little is known about its Mg2+ translocation and regulatory mechanisms.The total Mg2+ concentration through a negative feedback mechanism, where at low Mg2+ concentrations, unbinding of Mg2+ ions from the CorA soluble domain favors a series of conductive states, allowing Mg2+ permeation21. MRS2 and CorA only share a sequence identity of ~14%, so insight into MRS2 Mg2+ translocation and regulatory mechanism based on CorA has been limited. Until very recently, the only MRS2 structure reported is the monomeric N-terminal soluble domain of yeast MRS222, lacking the transmembrane domain. A recent study of the N-terminal domain of human MRS2 revealed it forms a dimer, contrasting the pentameric assembly of CorA23. However, the missing structural information of the MRS2 full-length protein including the pore region hampers the understanding of how Mg2+ translocation occurs through the pore and how MRS2 is regulated.MRS2 belongs to the CorA protein superfamily characterized by a highly conserved Glycine-Methionine-Asparagine (GMN) motif in the loop between transmembrane helix 1 and 2. In prokaryotes, CorA regulates the intracellular Mg2+. N-terminal sequencing of MRS2 revealed the mitochondrial transit peptide cleavage site at residue 71. From the homo-pentameric structures, M336 and R332 are identified as key gating residues, which are further tested by mutagenesis, and Mg2+-dependent growth and Ni2+-sensitivity assays. The structures also reveal that MRS2 possesses two Mg2+-binding motifs in the soluble domain between neighboring subunits, which are different from known bacterial CorA structures. Finally, an inter-subunit salt bridge between R116 and E291 in the soluble domain is found. Disruption of this salt bridge leads to an increase in channel activity. Altogether, this study provides insight into how translocation of Mg2+ is mediated via MRS2 and how it is regulated.Here, we use single particle cryo-EM to determine the structures of human MRS2 in the presence and absence of Mg2+ using affinity and size-exclusion chromatography  which suggested a cleavage at residue 50. Here, we used an experimental approach, N-terminal sequencing, of purified human MRS2 and the first amino acid detected is residue 71 starting with threonine  at its N-terminus, which is cleaved after translocation into the inner mitochondrial membrane. As such, we expressed human MRS2 conjugated with a C-terminal FLAG tag in Expi293F cells and purified it in the presence of Mg31 and archael32 members of this protein family and is contrary to the most recent prediction of human MRS2 to form a dimer23. The final 3D reconstruction of MRS2 in the presence of Mg2+ (referred to as MRS2-Mg2+) with C5 symmetry and without symmetry applied (C1) resulted in maps with average resolutions of 2.8 Å and 3.1 Å, respectively, with the highest local resolution estimated to be 2.3 Å   were identified along the pore and two  were found in between neighboring soluble domains in the MRS2-Mg2+ structures , closest to the mitochondrial intermembrane space is coordinated by the highly conserved GMN motif. Mg2+-1 interacts with a ring of backbone carbonyl oxygens of G360 and amine group of N362 with a distance of 3.8 Å and 4.3 Å, respectively, indicating that the Mg2+-1 is in hydrated form36. Mg2+-2, Mg2+-3 and Mg2+-4 are coordinated by a ring of hydroxyl oxygens of T346, carbonyl oxygens of N339, and carboxylic oxygens of D329 with a distance of 5.2 Å, 4.3 Å and 3.5 Å, respectively. A highly negatively charged surface was observed in the loop located at the intermembrane space and the soluble domain in the matrix facing the pore  to control Mg2+ flux40. Interestingly, R332 is conserved among eukaryotes and for example also found in yeast Mrs2 but not in prokaryotic CorA homologs  can only grow either by supplementing the growth medium with high Mg2+ concentrations, or by complementation with a functional Mg2+-transporter/channel. Although expression of the WT HsMRS2 and single gating mutants R332A or M336A is not sufficient to support the growth of the strain without the supplementation of Mg2+, double mutant R332A/M336A complements the growth synergistically  have been identified in the interface of neighboring subunits between their soluble N-terminal domains termed soluble sites 1 and 2  between subunit E and A of MjCorA is close to soluble site 1 in MRS2; however, it is coordinated by E155, Y186, T90 and Q62, which is different to the groups of negatively charged residues  in MRS2  with C5 symmetry and without symmetry applied (C1) were determined at an average resolution of 3.3 Å and 3.6 Å, respectively, with the highest local resolution estimated to be 2.7 Å  have been identified in the soluble domains of adjacent subunits30. Mg2+ ions are coordinated by D89 and D253 at the M1 site, and D253, E88, L12, D175 at the M2 site. It has been proposed that these M1 and M2 sites serve as divalent cation sensors responding to intracellular ion concentrations and modulating channel activation. Unbinding of Mg2+ in the low affinity M1 and M2 sites under low intracellular Mg2+ concentration facilitates channel opening. TmCorA D253K mutation abolished the Mg2+-dependent protease susceptibility28 and Mg2+-dependent channel inhibition20. Previous EPR, cryo-EM and MD-simulation studies of CorA show dramatic movements of the soluble domain under EDTA condition and suggest that the inter-subunit binding of Mg2+ in the soluble domain holds the five subunits in the closed, less flexible conformation44. In the present human MRS2-Mg2+ structure, two inter-subunit Mg2+ ions were also identified but these are in different positions when compared to those in CorA . In general, the reported structures45 are in agreement with ours, with 0.652 Å Cα RMSD between structures in the presence of Mg2+. Two Mg2+ ions are identified along the pore of the reported structures, corresponding to Mg2+−1, 4 in our study. Two additional Mg2+ ions  have been found in both of our structures, coordinated by a ring of hydroxyl oxygens of T346 and carbonyl oxygens of N339 with a distance of 5.2 Å and 4.3 Å, respectively. We reason that the additional two Mg2+ ions identified is likely due to higher Mg2+ concentration (40 mM) in our sample, compared to 20 mM used in the previous publication45. Interestingly, a tentative Cl− has been placed in the R332-ring which is proposed to facilitate Mg2+-permeation by masking the electrostatic repulsion caused by the R332-ring45. Besides, MD-simulation demonstrates that the presence of Cl− around the R332-ring lowers the energy barrier for Mg2+ permeation. When we built the MRS2-Mg2+ and MRS2-EDTA models based on our cryo-EM maps, we also see a consistent density above the R332-ring in both structures  under various conditions have been reported40, other proposed mechanisms of CorA channel opening include helix rotation of TMs displacing the hydrophobic gating residues46, as well as relaxation/bending motion of the soluble domains coupled with TM movement leading to gate opening39  following the protocol described previously47. Cells were harvested 48 h after transfection, and resuspended in Lysis buffer  supplemented with cOmplete, EDTA-free protease inhibitor cocktail (Roche). After cell disruption by sonication on ice, the membrane fraction was collected by ultracentrifugation at 100,000 × g for 1 h at 4 °C. The pelleted membranes were resuspended with Lysis buffer and pelleted again at 100,000 × g for 1 h. The membrane pellets were flash frozen in liquid nitrogen and stored at −80 °C until use. For solubilization, membranes at a protein concentration of ~4 mg/ml were solubilized with 1% (w:v) n-dodecyl-β-d-maltopyranoside (DDM) (Anatrace) and 0.1% cholesteryl hemisuccinate Tris salt (CHS) (Anatrace) for 2 h at 4 °C, followed by another round of ultracentrifugation to remove insoluble material. The supernatant was incubated with ANTI-FLAG M2 Affinity Gel (Millipore) on an end-over-end rotator for 2 h at 4 °C. The MRS2-bound beads were incubated in Lysis buffer containing 1% lauryl maltose-neopentyl glycol (LMNG) for 30 min at 4 °C, followed by extensive washing with Washing buffer (Lysis buffer supplemented with 0.01% LMNG). Proteins were eluted with 0.2 mg/ml 3× FLAG peptide. Eluted fractions were concentrated to ~3 mg/ml using a 50 kDa cut-off centrifugal filter and loaded onto a Superdex 200 Increase 3.2/300 size-exclusion column (Cytiva) with SEC buffer . Peak fractions (~0.5 mg/ml) were collected for cryo-EM grid preparation. For the MRS2 sample in 1 mM EDTA, the sample was prepared in the same procedure except that the sample was dialyzed overnight to remove MgCl2, followed by size-exclusion chromatography with 1 mM EDTA SEC buffer . The protein fractions were resolved and analyzed using 4–12% Bis-Tris gel (Invitrogen) for SDS-PAGE and 4–16% Bis-Tris gel (Invitrogen) for BN-PAGE.Full-length human MRS2 (Uniprot Q9HD23) conjugated with a FLAG tag at the C-terminus was expressed in Expi293F cells . Cells at a density of 2.0−3.0 × 10Purified MRS2 was resolved by SDS-PAGE, followed by transferring to methanol activated PVDF membrane in a XCell Mini-Cell Blot Module (Thermo Fisher) at 30 V for 2 h. The MRS2 band on PVDF was stained with Ponceau S, excised and shipped to Creative Proteomics for N-terminal sequencing via Edman degradation.48 at a nominal magnification of 25,000x, a pixel size of 3.04 Å/px, and defocus range between −1.5 µm and −2.5 µm. A total of 459 images was collected. Image processing was performed using cisTEM v1.0.049. 215,448 particles were picked from the micrographs after CTF estimation, extracted with a box of 108 px (~328 Å) and analyzed by 2D classification.3 µL of MRS2 (0.02 mg/mL) was applied on a glow discharged carbon-coated 400 square mesh copper grid (EMS) and incubate for 1 min. The grid was then blotted by filter paper (Whatman) and washed once with 3 µL of Nano-W negative staining solution (Nanoprobes) followed by incubation with 3 µL of Nano-W for 1 min. The grid was blotted to remove excess staining solution and air-dried by waving. Images were recorded using an FEI Tecnai T20 TEM operated at 200 kV with a direct electron detector K2 Summit (Gatan Inc). Data was collected using SerialEM3 μl purified MRS2 at approximately 0.5 mg/ml was applied to a glow-discharged 400-mesh R 1.2/1.3 Cu grid (Quantifoil). The cryo grids were blotted for 6 s at 4 °C and 95%, and plunge-frozen into liquid ethane using a Leica EM GP2 (Leica) and stored in liquid nitrogen. The grids were screened on an FEI Tecnai T20 TEM before data collection.48 using a Titan Krios  operated at 300 kV and equipped with an energy filter and K3 camera (Gatan Inc.). Movies of 50 frames with a dose of 1 e−/Å2 per frame  were recorded at a nominal magnification of 105,000x, corresponding to a physical pixel size of 0.83 Å/px (super-resolution pixel size 0.415 Å/px) in CDS mode at a dose rate of 10 e-/px/s and a defocus range of −0.7 to −2.0 µm. In total, 3991 and 9656 movies were collected for MRS2-Mg2+ and MRS2-EDTA, respectively . Particles were then subjected to 2D classification to remove junk particles. 573,010 good particles selected from one round of 2D classification was subjected to ab-initio reconstruction (K = 2) and heterogenous refinement (K = 2) with C1 symmetry to further sort particles. Non-uniform refinement was performed with 450,554 selected particles, followed by local motion correction and CTF refinement to correct for beam-tilt, spherical aberrations, and per-particle defocus parameters. Non-uniform refinement with polished particles resulted in maps at 2.8 Å (with C5 symmetry applied) and 3.1 Å (with C1 symmetry applied), according to gold-standard FSC = 0.143 criterion  was performed with the subset of particles after 2D classification, followed by heterogenous refinement (K = 3) using all particles with C1 symmetry was applied to remove junk particles. Particles from one class  were re-extracted at 1x binned pixel size of 0.83 Å and subjected to non-uniform refinement followed by another round of heterogenous refinement (K = 2) to further sort particles. Final non-uniform refinement by using 1,744,117 particles yielded maps at 3.3 Å (with C5 symmetry applied) and 3.6 Å (with C1 symmetry applied) according to gold-standard FSC = 0.143 criterion.2+ (C1 and C5) and MRS2-EDTA (C1 and C5) have been made available and have the following accession codes: EMD−41624, EMD-41629, EMD-41628 and EMD-41630, respectively.The summary of data collection and imaging processing parameters are shown in Supplementary Tables 2+, predicted MRS2 AlphaFold51 structure (AF-Q9HD23-F1) was used as an initial model. The initial model was rigid-body fitted into the local resolution filtered map using UCSF Chimera v.1.1652. The model was then manually rebuilt in COOT v.0.9.753 using the local resolution filtered map, which was generated from refinement with C5 applied. The Mg2+ ions assigned in the pore regions were confirmed in the C1 map. Loop regions  were built with the unsharpened map. Iterative rounds of manual refinement in COOT and real-space refinement in Phenix v.1.20.1-448754 were performed. For the MRS2-EDTA structures, models were generated from multiple rounds of manual refinement and real-space refinements with the final model of MRS2-Mg2+ as initial model. The quality of the model and fit to the density was assessed using MolProbity55 and Phenix54. All structural figures were prepared using UCSF Chimera v.1.1652 or UCSF ChimeraX v.1.456. PDBs have been made available together with the EM maps with the following PDB IDs: 8TUL (MRS2-Mg2+) and 8TUP (MRS2-EDTA).For the atomic model of MRS2-Mg6 cells/ml were transfected with the MRS2-GFP or MRS2(71-443)-GFP encoding pCMV plasmid using polyethylenimine (PEI) following the protocol described previously47. 24 h after transfection, the cells were stained with 100 nM MitoTracker Red (Thermo Fisher Scientific) or 1 µM ER-Tracker Red (Thermo Fisher Scientific) for 30 min at 37°C. The cells were washed twice with PBS and imaged using a Zeiss LSM 880 confocal laser scanning microscope.Expi293F cells at a density of 2.0–3.0 × 102+-auxotrophic E. coli strain , which has been used previously42, was transformed with His-TmCorA, MRS2(71-443)-FLAG, its mutants or without insert in a pET28a backbone. The transformants were inoculated and grown in kanamycin (50 µg/mL) containing LB medium (LBK) supplemented with 100 mM MgSO4, shaking at 250 rpm and 37 °C overnight. The overnight cultures were used to inoculate fresh LBK in a 1:100 ratio and grown until they reached an OD600 of 0.6. The bacterial cultures were then diluted 10-fold serially with LBK medium, spotted onto LBK agar plates containing 0.1 mM IPTG and 0 or 100 mM MgSO4, and incubated at 30 °C for 2 days. Plates were imaged using ChemiDoc MP Imaging System (Bio-rad).The MgTmCorA, MRS2(71-443)-FLAG, its mutants or without insert in a pET28a backbone. The transformants were inoculated and grown in LBK medium, shaking at 250 rpm and 37 °C overnight. The overnight cultures were used to inoculate fresh LBK in a 1:100 ratio and grown until they reached an OD600 of 0.6. The bacterial cultures were then diluted 10-fold serially with LBK medium, spotted onto LBK agar plates containing 0.1 mM IPTG, and 0 or 1.8 mM NiCl2, and incubated at 30 °C overnight. Plates were imaged using ChemiDoc MP Imaging System (Bio-rad).BL21(DE3)  was transformed with His-Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data