In recent years, a number of mass spectrometric and laser spectroscopic techniques have been developed and are increasingly used by the research community. Advances in this active research area, however, critically depend on the availability of suitable N2O isotope Reference Materials (RMs).Information on the isotopic composition of nitrous oxide  have been analysed by specialised laboratories against isotope reference materials. A particular focus was on the 15N site‐specific isotopic composition, as this measurand is both highly diagnostic for source appointment and challenging to analyse and link to existing scales.Within the project Metrology for Stable Isotope Reference Standards (SIRS), seven pure N2O isotope RMs offer a wide spread in delta (δ) values: δ15N: 0 to +104‰, δ18O: +39 to +155‰, and δ15NSP: −4 to +20‰. Conversion and uncertainty propagation of δ15N and δ18O to the Air‐N2 and VSMOW scales, respectively, provides robust estimates for δ15N(N2O) and δ18O(N2O), with overall uncertainties of about 0.05‰ and 0.15‰, respectively. For δ15NSP, an offset of >1.5‰ compared with earlier calibration approaches was detected, which should be revisited in the future.The established N2O isotope RMs anchored to the international isotope‐ratio scales was developed that will promote the implementation of the recommended two‐point calibration approach. Particularly, the availability of δ17O data for N2O RMs is expected to improve data quality/correction algorithms with respect to δ15NSP and δ15N analysis by mass spectrometry. We anticipate that the N2O isotope RMs will enhance compatibility between laboratories and accelerate research progress in this emerging field.A set of seven N For nitrogen, the 15N/14N isotope ratio is used, R(15N/14N) = x(15N)/x(14N), where x is the isotopic abundance and tropospheric N2 is the international reference material for the Air‐N2 scale. For oxygen, the 18O/16O and 17O/16O ratios are used, which are related to the Vienna Standard Mean Ocean Water (VSMOW) scale. In addition, we adopt the following notation conventions: δ15N = δ (average of both nitrogen atoms) and δ18O = δ. The 15N site preference (SP) is defined by the predominance of 15N substitution in the central (α) position as compared to the terminal (β) position, and calculated accordingly as δ15NSP = δ15Nα − δ15Nβ. All δ values in this paper are reported against Air‐N2 (for 15N/14N ratios) and against VSMOW (for 18O/16O and 17O/16O ratios).The isotopic composition of a sample is reported using the delta  as well as δ17O and δ18O (water) have a different chemical identity than N2O sample gas. Thus, a chemical conversion reactionFurther progress in N2O by thermal decomposition of isotopically characterised ammonium nitrate (NH4NO3) has been suggested as an approach to link the position‐dependent nitrogen isotopic composition of N2O to the Air‐N2 scale.α‐position of of the formed N2O originates from NO3−, while the β‐nitrogen comes from NH4+.4NO3 decomposition technique has been confirmed,δ15Nα and δ15Nβ was found to be limited by non‐quantitative NH4NO3 decomposition in combination with substantially different isotope enrichment factors of −4 or −19‰ for the conversion of the NO3− or NH4+ nitrogen atom into the α‐ or β‐position of the N2O molecule.2O reference gases, USGS51 and USGS52, recently became available with assigned δ values based on a preliminary assessment by Naohiro Yoshida and Sakae Toyoda (Tokyo Institute of Technology).δ15N and δ18O values (< 1‰), which is not suitable for a two‐point calibration approach.The synthesis of N2O RMs within the framework of the European Metrology Programme for Innovation and Research (EMPIR) 16ENV06 project ‘Metrology for Stable Isotope Reference Standards (SIRS)’. The target isotopic composition of N2O RMs was selected according to discussions at a stakeholder workshop at the 19th GGMT conference at Empa (29 August 2017).2O RMs compared to RMs presented in Ostrom et alδ17O data in order to improve data quality/correction algorithms with respect to δ15NSP and δ15N analysis by mass spectrometry. In addition, the link of δ values to the international isotope‐ratio scales was revisited.In the present study, we report the development of additional N22O RMs, covering an extended range of delta values as compared to existing gases. Figure 2O RMs.The main purpose of this study is the provision of isotopically characterised N15N/14N isotope ratios on the Air‐N2 scale were propagated from NH4+ and NO3− salts supplied by IAEA/USGS, through isotopic analysis of gravimetrically prepared NH4NO3 salts (section δ15Nβ(N2O) /δ15Nα(N2O) in the novel N2O RMs. The international RMs applied in this study are listed in Table 2O site‐specific isotopic composition, the NH4NO3 decomposition reaction was optimised for high yield, reproducibility, and N2O purity , δ18O(N2O), δ17O(N2O) and δ15NSP(N2O) is described. In one laboratory (Empa), δ15NSP(N2O) in the N2O RMs was linked to the Air‐N2 scale making use of the traceability chain established in section In section 2.12.1.14NO3 salts (S1–S6), covering a wide range of δ15N(NH4+) and δ15N(NO3−) values, were produced by gravimetric mixing of five commercially available NH4NO3 salts (A–E). A: unlabelled NH4NO3 , B: 15NH4NO3 , C: NH415NO3 , D: 15NH4+‐depleted NH4NO3 , E: 15NO3−‐depleted NH4NO3 .Six NH4NO3 salts (Table 4NO3 (A) was ground to a fine powder using a mortar and pestle and then dried at 120°C for 1 h (a temperature low enough to avoid triggering decomposition). From this, around 100 g (S1–S5) or around 40 g (S6) were gravimetrically  mixed with appropriate amounts of salts B, C, D, and E to obtain the desired isotopic composition. The salt mixtures were dissolved in deionised water , recrystallised, dried, and then stored in air‐tight sample containers. The isotopic homogeneity of S1–S6 was confirmed by repeated IRMS analysis (MPI‐BGC), demonstrating δ15N(NH4NO3) values within <0.2‰ .For preparation of these six NHts Table , approxi2.1.24NO3 salts (S1–S6) were sent together with international reference materials ((NH4)2SO4, NaNO3, KNO3) provided by the IAEA  and by USGS   Table .δ15N(NH4NO3), δ15N(NH4+) and δ15N(NO3−) results from all laboratories were calibrated using the provided international IAEA and USGS reference materials, with δ15N values and uncertainties according to Brand et alσcal) was estimated from the uncertainty  in the linear calibration function (Equation σmeas) in δ15Nmeas, following the law of error propagation  from individual laboratories i were combined to a weighted mean value  were weighed into round‐bottomed glass flasks with a break‐seal . In a variant of the NH4NO3 decomposition reaction according to Szabó et al,4HSO4  and 0.2 g (NH4)2SO4  were added. Adding surplus NH4+ salt will lead to a loss in δ15Nβ information but was included to test if very high reaction yields can be achieved, which might still be attractive. Therefore, for S1, both variants (with/without NH4HSO4/(NH4)2SO4) were tested, while for S2–S6 only decomposition without NH4+ addition was performed. Thereafter, the flasks were evacuated (<10−1 mbar) and flame‐sealed. The sealed flasks were placed in a circulating‐air oven  and heated to 270°C for 24 h.Aliquots of approximately 1.0 g (12.5 mmol) of NH2O product gas, e.g. S1‐derived‐N2O (here: S1‐N2O) or S6‐derived‐N2O (S6‐N2O), was purified on a vacuum manifold by cryogenic distillation. Reaction by‐ and side‐products  were trapped at −78°C (dry ice/ethanol bath); N2O was trapped at −196°C (liquid N2) in a coiled stainless‐steel tube, while N2 and O2 (side products) were removed by evacuation with an oil‐sealed rotary vane pump . Thereafter, the N2O product was condensed into 10 mL stainless‐steel flasks  under liquid‐nitrogen cooling. The cryogenic extraction was repeated five times to fully capture the produced N2O. Finally, the N2O yield was determined gravimetrically . The N2O purity, i.e. the absence of IR‐active impurities , was confirmed by FTIR spectroscopy .2 (<0.01%) and N2O recovery (>99.4%), using different gravimetric mixtures of high‐purity N2O and N2 .After the decomposition reaction, the N4NO3 decomposition reaction across the large range of δ values  was tested. In detail, such tests were made by comparing δ15Nα of NH4NO3‐derived N2O gases (S1‐N2O–S6‐N2O) with the δ15N(NO3−) of substrate NH4NO3 salts (S1–S6) and δ15Nβ with δ15N(NH4+), respectively. While the link provided by the NH4NO3 decomposition reaction was assumed to be valid across a wide range of δ values, the analytics involved in δ15Nα, δ15Nβ or δ15N(NO3−), δ15N(NH4+) analysis might display non‐linearities.First, the consistency of the NH2O gases S2‐N2O, S3‐N2O, S5‐N2O and S6‐N2O were analysed together with S1‐N2O and S4‐N2O using the QCLAS analyser  and in preliminary experiments displayed a consistent offset between δ15Nα(N2O), δ15Nβ(N2O) and δ15N(NO3−), δ15N(NH4+) values (data not shown). For actual δ15Nα and δ15Nβ of S1‐N2O and S4‐N2O, known δ15N(NO3−) and δ15N(NH4+) values of the respective NH4NO3 salts were adopted and no correction for fractionation effects due to incomplete decomposition or branching isotope effects due to N2 production was applied. The uncertainty of actual δ15Nα and δ15Nβ for S1‐N2O and S4‐N2O was estimated from the uncertainty of weighted mean δ15N(NO3−) and δ15N(NH4+) values  of the educt NH4NO3 salts S1/S4, were used to define a linear calibration function  was tested against the actual δ15Nα values, i.e. the δ15N(NO3−) of the respective NH4NO3 salts (S1–S6). The same procedure was applied to δ15Nβ and δ15N(NH4+).Measured 2.22.2.12O gases offer only limited isotopic variability. Therefore, high‐purity N2O  was supplemented with defined amounts of 15N‐enriched/15N‐depleted and 18O‐enriched N2O dopant gas using a ten‐port two‐position valve  with sample loops of different volumes , as well as 18O‐enriched N2O ((36.25 ± 0.10)% NN16O, (63.75 ± 0.76)% NN18O) and 15Nβ‐depleted N2O (δ15Nα = (−2.54 ± 0.005)‰, δ15Nβ = (−162.21 ± 0.03)‰, δ18O = (+38.92 ± 0.003)‰), both produced and characterised at Empa. Details on the production and analysis of 18O‐enriched N2O and 15Nβ‐depleted N2O are provided in the supporting information  or 150 mL (Lab MPI) stainless‐steel flasks  for isotopic analysis.The dopant gases were commercial ormation . N2O RMs2.2.2δ15Nα, δ15Nβ and δ18O in the N2O gases, a QCLAS spectrometer −1 and an astigmatic Herriott multi‐pass absorption cell (204 m path length) was applied. Prior to analysis, pure N2O gases  were diluted to around 50 μmol mol−1 using one cylinder of synthetic air ((20.5 ± 0.5)% O2 in N2, Messer Schweiz AG, Switzerland) into 2 L high‐pressure stainless‐steel cylinders  using a ten‐port two‐position valve . A singular cylinder of synthetic air was used for all experiments to minimise differences in the oxygen content, which would otherwise affect pressure broadening of absorption lines, result in differences in apparent isotopologue mole fractions and increase uncertainties. The selection of synthetic air as diluent is somewhat arbitrary and not meant to represent an alternative for a full‐air matrix for high‐accuracy ambient N2O isotope analysis, which would enclose noble and trace gases depending on the analytics and accuracy requirements.For analysis of δ15Nα = (+25.73 ± 0.24)‰, δ15Nβ = (+25.44 ± 0.36)‰, δ18O = (+35.86 ± 0.22)‰) and CG2 (δ15Nα = (−48.59 ± 0.25)‰, δ15Nβ = (−46.11 ± 0.43)‰, δ18O = (+27.37 ± 0.11)‰). The isotopic composition of the calibration gases had been previously analysed by Sakae Toyoda at the Tokyo Institute of Technology using their analytical technique as a link to the international scales.The spectroscopically determined isotope ratios were related to the isotope‐ratio scales realised by Toyoda et al2O RMs by QCLAS, the site‐specific isotopic information provided by NH4NO3‐derived N2O gases S1‐N2O (δ15Nα = (−1.41 ± 0.21)‰, δ15Nβ = (+0.33 ± 0.12)‰) and S4‐N2O (δ15Nα = (+52.36 ± 0.15)‰, δ15Nβ = (+53.06 ± 0.16)‰) was propagated to the N2O RMs (RM1–RM5). For this, the N2O RMs were analysed together with S1‐N2O and S4‐N2O, as described in the preceding section, to propagate the moiety‐specific isotopic composition defined by S1 and S4 to the novel RMs  MAT 252 mass spectrometer  against an isotopically characterised laboratory tank of pure N2O ; C1: δ15N = (−2.4 ± 0.4)‰, δ15Nα = (−4.5 ± 0.4)‰, δ15Nβ = (−0.3 ± 0.8)‰, δ18O = (+23.3 ± 1.2)‰. IRMS analysis of the N2O intramolecular 15N distribution was based on the quantification of the fragment NO+ (m/z 30 and 31) and molecular N2O+  ions to calculate isotope ratios for the entire molecule and the central (α) and terminal (β) N atom. Analysis of δ15N (45/44) and δ15Nα involves correction for interfering 14N217O+ (m/z 45) and 14N17O+ (m/z 31) using actual Δ17O values analysed at the University of East Anglia (UEA). For the analysis of δ15Nα and δ15Nβ, rearrangement of N atoms (Nα and Nβ) in the ion source was considered. The δ15N, δ15Nα and δ15Nβ values of the local reference gas were previously anchored to Air‐N2 by NH4NO3 decomposition,δ18O value was anchored to VSMOW by converting N2O into CO2 with graphite and a platinum foil . The analytical uncertainties were calculated from the uncertainty of the in‐house working N2O standard gases and the standard deviation for repeated measurements of the sample gas (N2O RM) and the in‐house working N2O standard following the law of error propagation. Specifically, the uncertainty of the in‐house working N2O standard gas for δ15N, δ15Nα and δ15Nβ values comprises both the uncertainty in the δ15N(NH4+) and δ15N(NO3−) analysis and the repeatability of the NH4NO3 decomposition reaction. For δ18O, the uncertainty of the in‐house working N2O standard gas includes the repeatability of the conversion reaction of N2O into CO2 with graphite. δ17O signatures of three N2O RMs  were analysed by high‐resolution IRMS . Experimental details of this prototype analyses are provided in the supporting information . The system and the method used have been described by Sperlich et al.δ15N values of the sample N2O were scaled to IAEA‐N‐1 and USGS32. In addition to the sample gases, an in‐house standard N2O gas NINO was analysed in each sample run, which was used as an anchor for δ15N measurements by DI‐IRMS. USGS40, and the in‐house standards Ali‐j3 (δ15N = (−1.51 ± 0.1)‰; acetic anilide) and Caf‐j3 (δ15N = (−15.46 ± 0.1)‰; caffeine), were analysed in each daily run as quality controls, but not used for data correction.2O RMs were analysed twice  on a DI‐IRMS system  using separately subsampled flasks. We note that the published δ values for USGS51 and USGS52 are average values with a rather large deviation between laboratories. Therefore, we scaled the DI‐IRMS δ15N analyses to the in‐house standard NINO using the value reported for the primary calibration using EA/IRMS (δ15N = (+0.54 ± 0.21)‰). The δ18O value of NINO (δ18O = (+39.94 ± 0.34)‰) was determined by setting the δ18O of USGS51 equal to the average value from the interlaboratory comparison (δ18O = (+41.45 ± 0.34)‰) published by Ostrom et al.The Nδ15N is measured from N2 gas, the DI‐IRMS method allows the analyses of δ15N and δ18O values by simultaneously recording m/z 44 (14N14N16O+), 45  and 46  ion currents. δ15N and δ18O values for N2O RMs were calculated according to Kaiser et al17O values determined by UEA were used.In contrast to the EA/IRMS technique, where δ15N and δ18O measurements of the N2O standard gases  and from the standard deviation for repeated measurements of the sample gas (N2O RM) and the N2O standards, following the law of error propagation.The uncertainty of the analyses was calculated from the uncertainty of 2.2.52O RM samples and a N2O‐MG‐6.0 working reference  were diluted to 0.09 mmol mol−1 in N2 , filled into 20 mL serum vials  and analysed for δ(Ν2Ο) and δ(Ν2Ο) on a custom‐built automated cryogenic extraction and purification system comprised of an autosampler, a valve system, and PoraPLOT Q pre‐ and main columns , coupled to a GEO 20‐20 isotope ratio mass spectrometer .The Nδ(O2) = δ17O, δ(O2) ≈ δ18O (the error of this approximation is <0.01‰) and 29δ(Ν2) = δ15Ν after cryogenic N2O extraction and decomposition to N2 and O2 with a 500 mm long pure gold tube  held at 854°C. N2 and O2 were separated directly (without further cryofocussing) on a molecular‐sieve 5‐Å PLOT main column ). The quantitative conversion of N2O was verified by swapping the molecular‐sieve main column for the PoraPLOT Q main column and testing for residual N2O with the mass spectrometer. The raw δ17O and δ18O measurements were affected by scale compression. To correct for this, a logarithmic scale normalisationδ18O value of +112.4‰ (relative to N2O‐MG‐6.0) derived from the δ(Ν2Ο) measurements of the diluted RM4 sample measured on the GEO 20‐20 mass spectrometer. The same normalisation was used for δ17O as for δ18O because no N2O reference material with a calibrated δ17O value was available. No scale‐normalisation was applied to the δ15N measurements. Uncertainties were calculated using the law of error propagation from the standard deviations of replicate measurements against the working reference gas and the calibration uncertainties of the working reference gas against Air‐N2 and VSMOW.Using the same mass spectrometer, these samples were also analysed for 2O RM samples were analysed for δ(Ν2Ο) and δ(Ν2Ο) with respect to the N2O working reference N2O‐MG‐6.0 using the dual‐inlet system of a Finnigan MAT 253 isotope ratio mass spectrometer . The N2O‐MG‐6.0 working reference has been calibrated by Kaiser et al,δ15N = (+1.01 ± 0.06)‰ with respect to Air‐N2, as well as δ18O = (+38.45 ± 0.22)‰ and δ17O = (+19.66 ± 0.11)‰ with respect to VSMOW.δ17O values of N2O RMs analysed with the Sercon GEO 20‐20 were used for the data correction according to Kaiser et al.2 and VSMOW.The N33.1δ15Nα and δ15Nβ in N2O to the Air‐N2 scale and calculating uncertainties are described. Section δ15N(NH4+) and δ15N(NO3−) analyses in NH4NO3 salts (S1–S6) by eight isotope laboratories against international IAEA and USGS standards. Section 4NO3 decomposition at high yield, repeatability, and N2O purity. To enable the two‐point calibration, a number of NH4NO3 salts with different isotopic composition were produced and decomposed and the consistency of δ15Nα and δ15Nβ of the N2O gases (S1‐N2O–S6‐N2O) and the δ15N(NH4+) and δ15N(NO3−) of NH4NO3 salts (S1–S6) was tested , as analysed by the eight isotope laboratories and calibrated to Air‐N2 by analysis of IAEA and USGS standards, is indicated in Table 4NO3 samples , all results obtained by EA/IRMS were included for calculation of the weighted mean value except for results by one laboratory (Lab 4), as this laboratory used a more complicated analytical procedure with higher uncertainties. For δ15N(NO3−) and δ15N(NH4+), all laboratory results were included to calculate the weighted mean values, irrespective of the applied analytical technique.For δ15N(NH4NO3) with average δ15N(NH4+)/δ15N(NO3−) values indicates a good agreement to within <0.2‰. Nonetheless, results by individual laboratories for moiety‐specific δ values deviate substantially from the weighted mean. As an example, δ15N(NO3−) results from Lab 8 are substantially higher than those from other laboratories by an average of (+2.15 ± 0.58)‰. This may be due to the specific preparation technique applied, NH4+ removal by ion exchange, a technique which is prone to preferential retention/elution of 15NO3.δ15N values of both NO3− and NH4+, which may be reflected in the δ15N(NH4+) values of Lab 6 but not those of Lab 5, where a similar technique was used. Conversely, systematic fractionation effects by preparation techniques should be accounted for by identical treatment (IT) of the provided IAEA and USGS standards used for data correction. In summary, analysis of δ15N(NH4+) and δ15N(NO3−) is still challenging; however, the ensemble of techniques applied in this study provides good agreement with δ15N(NH4NO3) values.A comparison of 3.1.22O yield of 93–95% was achieved for the decomposition of NH4NO3 salts S1–S6 (Table n = 3), surpasses results by Westley et aln = 20). A further increase in the yield of the NH4NO3 decomposition was achieved by conducting the reaction in a NH4HSO4–(NH4)2SO4 melt (around 2%), as suggested for industrial applications by Szabó et alδ15Nα values but a loss in the δ15Nβ information due to NH4+ salt addition, and was thus not continued.Under optimised reaction conditions  and distillation procedure, an average Nδ15Nα and δ15Nβ for the loss in N2O (around 5–7%), mainly due to uncertainties in the reaction mechanisms (incomplete decomposition or side‐reaction), which makes it difficult to estimate the effect on δ values. Assuming incomplete reaction accompanied by fractionation effects, according to our earlier study,δ15Nα/δ15Nβ/δ15N values, respectively. However, a much smaller difference in δ15N was observed when comparing results of N2O RMs analysed by QCLAS  with IRMS analyses. Therefore, our assumption is that the decrease in yield is at least partly caused by a “branching” side reaction, e.g. nitrogen gas (N2) production,δ15N(N2) values.2 production has a minor effect on δ15Nα, δ15Nβ and δ15NSP, but the effect is expected to depend on the timing of N2 generation, which is not known.No correction was applied to 3.1.32 scale and to determine the N2O site‐specific isotopic composition across a wide range of δ values. Therefore, the consistency of the isotopic composition of the N2O gases  and the NH4NO3 salts (δ15N(NH4+) and δ15N(NO3−), S1–S6) was tested. The detailed procedure is described in section 4NO3 decomposition reaction, measured δ15Nα values of S1‐N2O/S4‐N2O and actual δ15Nα values, i.e. δ15N(NO3−) of the educt NH4NO3 salts S1/S4, were used to define a linear calibration function. δ15Nαcal values of S2‐N2O, S3‐N2O, S5‐N2O and S6‐N2O were calculated from measured δ15Nα using this correction function and compared against actual values /δ15N(NH4+)/δ15N(NO3−) – δ15N(NH4+) of the respective salts . Assuming similar fractionation effects for decomposition of all NH4NO3 salts (S1–S6), provided the comparable decomposition yield  and δ15N(NH4+) analyses of the NH4NO3 salts. The latter is more plausible, as the QCLAS analyses using the same calibration approach showed good agreement with independent IRMS measurements for N2O RM with high 15N enrichment  (e.g. S5), which agrees with earlier studies indicating challenges in δ15N(NH4+) analysis, but this may also be due to the lack of available international standards for δ15N(NH4+) that cover δ values above (+53.75 ± 0.24)‰ (USGS26) and below (−30.41 ± 0.27)‰ (USGS25).Results of O3 salts –S6, prov2O gases from around zero (S1‐N2O) to 15N‐enriched (S4‐N2O) and of the substrate NH4NO3 salts (S1–S4). Thereby, our study covers a much larger range of δ values  than earlier studies,2 scale. At very high and low 15N enrichment , the calibration approach using NH4NO3 decomposition is more challenging, probably due to less satisfying analytical accuracy of δ15N(NH4+) measurements to date. As the N2O gases S5‐N2O and S6‐N2O were not included in the analysis of N2O RMs, their enhanced uncertainty in δ15Nαcal and δ15Nβcal does not affect the data quality of N2O RMs.In summary, our results demonstrate consistency of the isotopic composition of the N3.23.2.12O RMs (RM1–RM5) were calibrated against Air‐N2 by both QCLAS (Lab Empa) and IRMS (Lab TT) analyses. For QCLAS analyses, two N2O gases produced by NH4NO3 decomposition  were applied to define a calibration function and propagate the isotopic information of the NH4NO3 salts (δ15N(NO3−), δ15N(NH4+)) to the N2O RMs . δ15NSP and δ15N were calculated using definitions and their uncertainty estimated using the law of error propagation. In Table δ15NSP values acquired by QCLAS (Lab Empa) using the calibration approach established in this study are compared with results provided by DI‐IRMS (Lab TT) using a previously published link to the Air‐N2 scale.2O RMs are shown in Tables The novel Nδ15NSP measurements by DI‐IRMS  and QCLAS (Lab Empa) across all N2O RMs. This is most likely attributable to the calibration of the position‐dependent δ values with respect to Air‐N2 via the NH4NO3 decomposition technique, which were performed independently for the two labs. Incidentally, for the NH4NO3 salts S1–S4, the δ15N(NO3−) results provided by Lab TT were always lower ((−0.63 ± 0.59)‰), while δ15N(NH4+) values were higher than the respective weighted mean values ((+0.49 ± 0.25)‰), which would lead to 1.12‰ lower δ15NSP values ‰ vs. (+18.7 ± 2.2)‰ for ambient tropospheric N2O.δ15NSP measurements by the Tokyo Institute of Technology, using the NH4NO3 decomposition technique.A similar 1.5–2.0‰ difference in δ15NSP and the Air‐N2 scale with high accuracy is still challenging and the current realisation of the Air‐N2 scale for δ15NSP provided by USGS51 and USGS52δ15NSP values and should be revisited in future studies.We conclude that the realisation of the link between 3.2.2δ15N values of N2O RMs were analysed by IRMS in three different laboratories using independent links to the Air‐N2 scale ‰ (USGS51) and (+0.07 ± 0.22)‰ (USGS52). These values fall between results published by laboratories 7 and 8 (USGS and BGC‐IsoLab) in Ostrom et al,δ15N(N2O) measurements. A similar offset between laboratories had already been detected earlier and was attributed to differences in the propagation of the Air‐N2 scale to δ15N(N2O).2O RMs.le Table . Results52 Table  by a com17O data are available. Consistency of N2O RM flask subsamples was demonstrated using DI‐IRMS  and 0.03‰ for δ18O(N2O) .In contrast, differences between analytical techniques applied within one lab, thus using the same link to the scale, were smaller than offsets between laboratories: (+0.10 ± 0.04)‰ for Lab MPI and (+0.12 ± 0.14)‰ for Lab UEA. This indicates that both EA/IRMS and GC‐IRMS and DI‐IRMS can achieve high accuracy, provided that an accurate link to the scale and Δ3.2.3δ18O values of N2O RMs were analysed by IRMS in three different laboratories , indicating a potential scaling or scale compression issue. Measurements were not completely independent for all laboratories, as the results for Lab MPI‐II were referenced to average δ18O values of USGS51 in Ostrom et al,es Table . Resultsδ17O values were determined by GC/IRMS at UEA (Lab UEA‐I) and showed a (0.98 ± 0.27)‰ offset to prototypical measurements by HR‐IRMS (MAT253 ULTRA) at the Tokyo Institute of Technology 2SO4 melt. n indicates the number of decomposition experiments.Table S2. Isotopic composition of RMs, analysed by QCLAS at Empa versus S1‐N2O and S4‐N2O to calculate δ15Nα, δ15Nβ, δ15NSP, and δ15N values. n indicates the number of analyses. Uncertainties are calculated using the law of error propagation involving the uncertainties in S1‐N2O and S4‐N2O, their analyses, and the analyses of N2O RMs but do not enclose deviations due to fractionation or branching effects during NH4NO3 decomposition. All values are reported in ‰.Table S3. Isotopic composition of RMs, analysed by IRMS at Tokyo Institute of Technology (Lab TT) versus an isotopically characterised in‐house working standard to calculate δ15Nα, δ15Nβ, δ15NSP, δ15N, and δ18O values on the “Tokyo Tech scale”. n indicates the number of analyses. Uncertainties are calculated using the law of error propagation. All values are reported in ‰.Table S4.δ15N of RMs, the in‐house N2O standard gas (NINO), and a number of quality control standards, analysed by Lab MPI  versus primary reference materials and second scale anchor of the Air‐N2 scale . n indicates the number of analyses. Expanded uncertainties are calculated following the law of error propagation. For the quality control, standards target values and references are provided as well. All values are reported in ‰.Table S5. Isotopic composition of RMs, analysed as N2O diluted to 0.09 mmol mol−1 on Sercon GEO 20–20 IRMS (UEA‐I) after gold decomposition, scale‐normalised to the δ18O value of RM4. n indicates the number of analyses. Uncertainties are calculated using the law of error propagation from the standard deviations of replicate measurements against the working reference gas and the calibration uncertainties of the working reference gas against Air‐N2 and VSMOW.14 All values are reported in ‰.Table S6. Isotopic composition of RMs, analysed as pure N2O on Finnigan MAT 253 IRMS (UEA‐II) using the actual Δ17O measurements. n indicates the number of analyses. Uncertainties are calculated using the law of error propagation from the standard deviations of replicate measurements against the working reference gas and the calibration uncertainties of the working reference gas against Air‐N2 and VSMOW.15 All values are reported in ‰.Click here for additional data file.


The main difference between molecules A and B involves the dihedral angles of the phenyl groups. One phenyl ring makes dihedral angles of 71.14 (6)° (molecule A) and 82.81 (7)° (molecule B) with respect to the core (C4N3O2) of the molecule [14.56 (9)° (molecule A) and 5.7 (1)° (molecule B) for the other phenyl ring]. Another prominent feature is the intramolecular N—H⋯O hydrogen bond present in both crystallographically independent molecules.The title compound, C E)-N-Phenyl-N-(phenylcarbamoyl)-3-[propyl(trimethylsilyl)amino]acrylamide is an insertion product from propyl(trimethylsilyl)[2-(trimethylsilyl)ethenyl]amine and phenyl isocyanate. It was obtained in the course of our work on different types of silicon–nitrogen compounds  carbamoyl]-3-(methylsulfanyl)acrylamide  Å in molecule B] The planarity is presumably due to the conjugated system of double bonds. The C14–C19 phenyl rings in both molecules are not coplanar to the core of the molecules but adopt dihedral angles to the latter of 14.56 (9)° (molecule A) and 5.7 (1)° (molecule B). This small deviation from planarity still allows conjugation between the C14–C19 phenyl ring and the urea part of the molecule.The core of the molecule formed by N1, C4–C6, O1, N2, C7, O2 and N3 is almost planar in both molecules of the title compound [the average deviation from the plane is 0.05 (6) Å in molecule A) and 82.81 (7)° (molecule B) with the core of the molecule. This almost perpendicular conformation may be explained by the presence of the oxygen atom O2 in a vicinal position to the respective phenyl group.The C8–C13 phenyl rings in both molecules subtend dihedral angles of 71.14 (6)° -N-phenyl-N-(phenylcarbamoyl)-3-[propyl(trimethylsilyl)amino])acrylamide was obtained from the reaction of propyl(trimethylsilyl)[2–(trimethylsilyl)ethenyl]amine and phenyl isocyanate. As shown in Fig. 4et al., 2018(n-pentane were added dropwise 0.35 g (3 mmol) phenylisocyanate at 0°C. After standing for six days at room temperature, volatiles were removed under reduced pressure. Storing the product mixture for five years at −28°C yielded crystals suitable for single-crystal X-ray diffraction. No quantitative yield can be given here, since only a few crystals at the wall of the Schlenk tube were available. NMR spectroscopy showed that the batch product is a mixture of many components. Further purification of the product mixture was not successful.To a solution of 0.46 g (2 mmol) propyl(trimethylsilyl)[2-(trimethylsilyl)ethenyl]amine in 10 ml Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623001177/im4017sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623001177/im4017Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623001177/im4017Isup3.cmlSupporting information file. DOI: 2240673CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structure of the title compound has been characterized by single-crystal X-ray diffraction and Hirshfeld surface analyses. The molecular structure and frontier orbitals were also investigated using DFT. 11H9BrN2O, is almost planar. The benzene and pyrimidine rings are essentially coplanar, with r.m.s. deviations of 0.0130 Å, and the largest displacement is for the flap atom of the dihydropyrrole moiety [0.154 (7) Å]. Hirshfeld surface analyses revealed that the crystal packing is dominated by H⋯H, Br⋯H/H⋯Br and O⋯H/H⋯O interactions, and Br⋯Br interactions in the crystal structure are also observed. Theoretical calculations using density functional theory (DFT) with the B3LYP functional basis set gave numerical parameters for the frontier molecular orbitals.The molecular structure of the title compound, C In comparison with a reported literature procedure  at room temperature (293–298 K) and the reaction products separated by extraction after the reaction mixture was reduced to pH = 9–10 with NH4OH. As distinguished from the reported procedure, we carried out these reactions by cooling in an ice bath at a much lower temperature (273–275 K) and for a relatively longer period of time. The reaction products were finally separated by cold NH4OH at pH = 10–11. In general, the interactions of 7-bromo-2,3-dihydropyrroloquinazolin-9(1H)-one with aldehydes are well-studied  with the B3LYP functional basis set.2.et al., 2009et al., 2007aet al., 1992et al., 2009et al., 2005The molecular structure of the title compound is shown in Fig. 13.et al., 1964Cg interactions, with Br⋯Cg1 = 3.6428 (15) Å, forming a layered network  Å  and a ring slippage of 1.569 Å, and Cg2⋯Cg3ii being 3.7513 (16) Å  and a ring slippage of 1.194 Å. Both short intermolecular contacts help to stack parallel molecules along [100]. The resulting two-dimensional network extends parallel to (002), with neighbouring layers linked through C1—H1B⋯N4 short intermolecular contacts, H1B⋯N4 = 2.73 Å, C1—H1B⋯N4 = 169°, to form the full three-dimensional structure  Å, which is shorter than the sum of van der Waals radii  to the Hirshfeld surface . Besides these contacts, Br⋯H/H⋯Br (19.6%), O⋯H/H⋯O (11.3%), N⋯H/H⋯N (8.1%) and C⋯H/H⋯C (6.9%) interactions contribute significantly to the total Hirshfeld surface; their decomposed fingerprint plots are shown in Fig. 6c–f. The contributions of further contacts are only minor and amount to N⋯C/C⋯N (3.5%), O⋯C/C⋯O (2.0%), Br⋯C/C⋯Br (0.9%), Br⋯Br (0.8%), O⋯N/N⋯O (0.5%) and Br⋯N/N⋯Br (0.3%).In order to quantify the intermolecular interactions in the crystal of the title compound, a Hirshfeld surface (HS) analysis , chemical hardness (h) and chemical softness (s) can be calculated as follows: c = (I + A)/2; h = (I - A)/2; s = 1/2h, where I and A are the ionization potential and electron affinity, respectively, where I = –EHOMO and A = –ELUMO , hardness (h), potential (m), electrophilicity (w) and softness (s) for the title molecule were calculated at the DFT/B3LYP level using the 6-311++G basis set  indicates the relatively high stability of the title molecule.t Table 1. The val6.et al., 2016b]quinazolin-9(1H)-one moiety with a similar conformation to that in the title structure: deoxyvasicinone -one moiety gave only two hits: N-propanamide sesquihydrate -one monohydrate  (0.142 mol) of phosphoroxychloride were added dropwise over 1 h at 273–275 K. The reaction mixture was then heated at 368–371 K for 2 h, it was subsequently cooled and finally poured over ice. The temperature of the mixture was kept at around 273–275 K. When the reaction mixture was completely decomposed, it was brought to pH = 10–11 with 25%wt ammonium hydroxide solution. The light-yellow precipitate was filtered off, dried and recrystallized from methanol. The yield of the product was 4.35 g (82%), m.p. 431–433 K : 8.4 , 7.8 , 7.5 , 4.2 , 3.18 , 2.31 .8.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022007800/wm5655sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022007800/wm5655Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022007800/wm5655Isup3.cmlSupporting information file. DOI: 2194365CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal of the title flavone derivative, O⋯I halogen bonding and T–shaped π–stacking combine to generate a herringbone packing motif. 19H13IO2, the dihedral angle between the naphthyl ring system and the pendant iodophenyl ring is 72.48 (11)°. In the crystal, C—H⋯π interactions and I⋯O [3.293 (2) Å] halogen bonds are observed, which combine to generate a herringbone packing motif.In the title compound, C Our group is particularly interested in the extended flavonol motif as it has been shown to release CO quantitatively with visible light (Popova Cg1⋯Cg2i = 4.929 (2) Å  and C6—H6⋯Cg2i = 154.5 (3)°, where Cg1 is the centroid of the pyranone ring containing atoms C4–C7/C12/C13 and Cg2 is the centroid of the iodophenyl ring containing atoms C14–C19  Å, with C17—I1⋯O2ii and I1⋯O2ii—C1ii angles of 177.21 (10) and 127.9 (2)°, respectively . This I⋯O separation is some 0.25 Å shorter than van der Waals’interaction distance of 3.5 Å  was added and the reaction was stirred until a precipitate formed. The reaction mixture was acidified to pH 4 with glacial acetic acid. The solids were filtered and taken directly to the next step. (E)-1--3-(4-iodophenyl)prop-2-en-1-one was then suspended in ethanol (10 ml). An NaOH solution  was added and the reaction stirred until a precipitate formed. The reaction mixture was acidified to pH 1 with HCl (6 M). The white solid was collected by filtration and slow evaporation of a solution of the title compound in ethyl acetate gave colorless crystals .1-Acetyl-2-naphthol  and 4-iodobenzaldehyde  were dissolved in ethanol (5 ml). An NaOH solution  δ = 9.46 , 7.95 , 7.80–7.75 , 7.65 , 7.44 , 7.26 , 7.16 , 5.54 , 3.16 , 2.95  ppm.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2414314620001108/hb4334sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314620001108/hb4334Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620001108/hb4334Isup3.cmlSupporting information file. DOI: 1980111CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The TaV atom has a distorted tetrahedral coordination environment in a three-legged piano-stool fashion. The conformation of the pentafulvene exocyclic C atom to the three other ligands is staggered and not eclipsed, as found in the crystal structure of 1. Intermolecular interactions include π–π stacking, H⋯π interactions and weak C—H⋯Cl hydrogen bonds.The reaction of (η The C6exo atom coordinates roughly opposite of Cl2 to the central tantalum atom [C6—Ta1—Cl2: 171.58 (3)°]. Relative to the centroid of the five-membered ring (Ct), the angles to the chloride ligands are smaller than to the nitrogen ligands . The bond length Ta1—N1 [2.0433 (9) Å] and the sum of angles at N1 [347.1 (2)°] indicates a weak interaction of the nitrogen lone pair with the metal. The pentafulvene coordinates in a π-η5:σ-η1 fashion and exhibits typical distortion parameters . The C—C bond lengths within the pentafulvene are summarized in Fig. 2b. The pentafulvene has a ring slippage Δ of 0.31 Å and a θ angle of the Cipso—Cexo bond out of the plane of the five-membered ring of 36.30 (12)°. The Cipso—Cexo bond is a single to double bond . Consequently, the Ta1—Cl2 bond [2.3965 (3) Å) is longer than the Ta1—Cl1 bond [2.3452 (3) Å]. These pairs form a double-chain , linked by supramolecular contacts of the pentafulvene and the carbazolide ligands via π–π stacking [C1⋯C17: 3.3867 (15) Å] and an H⋯π interaction [C15⋯H10c: 2.773 (6) Å]. Numerical details of other hydrogen-bonding interactions are summerized in Table 1On the supramolecular level, around an inversion center, two molecules mutually interact in Fig. 3b, linke1 was prepared according to Riley et al.  was dissolved in tetrahydrofuran (20 ml) and cooled to 223 K. One equivalent of etheric HCl  was added dropwise and the solution was slowly brought to room temperature. After stirring over night, the solvents were removed in vacuo and the residue was extracted with toluene (10 ml). The solution was diluted with n-hexane (10 ml) and stored at 277 K for three days to yield a red crystalline material containing 1 and 2 (1:1). 1H NMR : δ = 0.79 , 0.85 , 1.27 , 1.49 , 1.53 , 2.13 , 2.62 , 3.27 , 3.65 , 6.30–8.29  p.p.m.Complex SHELXL . Refinement with OLEX2 . Refining all atoms anharmonically was dismissed, because it lowers the reliability factors only marginally, but more than triples the refinement parameters (263 versus 888 parameters).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314622012019/wm4176sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314622012019/wm4176Isup2.hklStructure factors: contains datablock(s) I. DOI: 2231842CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The Cp rings in both ferrocene groupings are close to eclipsed. In the crystal, O—H⋯O hydrogen bonds link the molecules into [100] chains.In the title compound, [CoFe The resulting complexes may have applications in the fields of electroluminescent devices, environmental sensors, photodynamic therapy and biological imaging °. The Cp rings of both ferrocene groups are in nearly eclipsed conformations.The cobalt(II) atom has a octahedral coordination environment defined by two trifluoromethyl-β-diketone ferrocene ligands and two water ligands Fig. 1 with thex, 1 − y, 1 − z) contact .In the crystal, the molecules are linked into [100] chains by O—H⋯O hydrogen bonds arising from the water molecules Table 1; an intrIn a 250 ml round-bottom flask, trifluoromethyl-β-diketone ferrocene (0.52 g 1.6 mmol), triethylamie (0.25 g 2.45 mmol) and cobalt acetate (0.13 g 0.5 mmol) were dissolved in 100 ml of methanol and the mixture was stirred at 343 K for 12 h and then cooled to room temperature. A red solid was obtained by suction filtration. Crystals for X-ray analysis were obtained by recrystallization from methanol solution.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620012407/hb4360sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314620012407/hb4360Isup3.hklStructure factors: contains datablock(s) I. DOI: 2031062CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A series of O—H⋯N interactions connect the molecules into twisting wires, which are cross-linked through van der Waals interactions.A co-crystal consisting of a 1:1 ratio of  p-vanillic acid (1/1)], C12H10N2·C8H8O4, the dihedral angle between the pyridine rings is 59.51 (5)°. In the crystal, the molecules are linked by O—H⋯N hydrogen bonds, generating [401] chains of alternating C12H10N2 and C8H8O4 molecules.In the title 1:1 co-crystal [alternatively called bipyridine ethylene– The vanillic acid has two distinct O—H⋯N-type hydrogen-bonding interactions (Table 1para-position hydroxyl group and the other pyridine N atom of a BPyE molecule resulting in a 2.6868 (13) Å distance between heteroatoms  was added to a 25 ml scintillation vial to which methanol was added until both compounds dissolved (approximately 20 ml). The resulting solution was vortexed for 30 s at 3000 rpm on a VWR Mini Vortexer MV I. The solution was then stored in the dark uncapped to allow for crystal formation while the solvent slowly evaporated.A 1:1 molar ratio of bipyridine ethylene  and Crystal data, data collection, and structure refinement details are summarized in Table 210.1107/S2414314622003042/hb4402sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314622003042/hb4402Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314622003042/hb4402Isup3.cmlSupporting information file. DOI: 2160226CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structure exhibits disorder of one of the 4-methoxybenzyl groups, the hydroxy group bonded to the 1,3-diazinan ring, and the methyl group of the methoxy residue. The crystal packing is sustained by C—H⋯O and O—H⋯π interactions, giving rise to infinite chains running along the b-axis direction.The title compound resulted from the condensation reaction between 1,3-bis{[(4-methoxyphenyl)methyl]amino}propan-2-ol and 2-hydroxybenzaldehyde in CH 26H30N2O4, at 173 K has orthorhombic (Pbca) symmetry. It was previously described by Bolte et al. . The title compound resulted from the condensation reaction between 1,3-bis{[(4-methoxyphenyl)methyl]amino}propan-2-ol and 2-hydroxybenzaldehyde in CH3OH. The structure exhibits disorder. One of the 4-methoxybenzyl groups, the hydroxy group bonded to the 1,3-diazinan ring, and the methyl group of the methoxy residue are disordered over two orientations, with occupancies of 0.807 (3)/0.193 (3), 0.642 (5)/0.358 (5), and 0.82 (4)/0.18 (4), respectively. The dihedral angles between the mean planes of the central 1,3-diazinan-5-ol and the 4-methoxyphenyl rings (both occupancy components of the disordered ring) are 88.65 (13), 85.79 (14) and 83.4 (7)°. The crystal packing is sustained by C—H⋯O and O—H⋯π interactions, giving rise to infinite chains running along the b-axis direction.The redetermined structure of 2-(2-hydroxyphenyl)-1,3-bis(4-methoxybenzyl)-1,3-diazinan-5-ol, C It is noteworthy that the coupling constants with magnitudes between 2.9 and 3.1 Hz provide a strong evidence of the presence of an axial OH group. In this regard, it has been reported that the presence of an intramolecular hydrogen bond may stabilize the hydroxyl group in the otherwise non-preferred axial position methyl]amino}propan-2-ol, easily obtained following the reported method  Å, θ = 3.1 (3)°, φ = 250 (4)°, Q(2) = 0.035 (3) Å and Q(3) = −0.561 (3) Å. Atoms C2 and C3 deviate from the mean plane of the other four atoms by −0.242 (3) and 0.249 (3) Å, respectively. Atoms N1 and N2 are essentially tetrahedral (bond-angle sums are 331.8° for N1 and 330.1° for N2), while the usual ΣH–N–H angle in an ammonia molecule is 321°  Å] and the C21–C26, C31–C36 and C31′–C36′ phenyl rings of the 1,3- benzyl groups are 88.65 (13), 85.79 (19) and 83.4 (7)°, respectively, whereas the mean plane is rotated by 81.22 (13)° towards the C11–C16 phenyl ring of the 2-hydroxyphenyl substituent. The dihedral angles between this phenyl ring and the other two phenyl rings are 55.54 (13)° (C21–C26), 84.27 (19)° (C31–C36) and 77.9 (7)° (C31′–C36′), respectively.S(6) graph-set motif (Table1). The N⋯O distance [2.740 (3) Å] is long in comparison with the values observed in related structures  is shorter than 4 Å. It is noteworthy that the interplanar distance between the symmetry-related main parts of the C31–C36 ring is only 3.66 Å; however, the corresponding Cg⋯Cg distance is too long at 4.619 (3) Å, indicating a significant horizontal shift of the rings precluding π–π stacking.In the crystal structure, the molecules are interlinked ds Fig. 3 into chas Table 1 amino}propan-2-ol  in methanol (20 mL) salicylaldehyde  was added dropwise. The resulting mixture was heated at reflux for 2 h and allowed to cool to room temperature. The solvent was removed under vacuum and the crude solid was washed with cold methanol and dried 1H NMR  δ 7.15 , 7.12–7.13 , 7.11 , 6.99 , 6.85 , 6.81 , 3.88 , 3.76 , 3.72–3.74 , 3.03 , 2.99 , 2.98 , 2.22 , 1.60 . The hydrogen atom of a hydroxyl group could not be assigned because of the overlaping and widening of that signal with those due to hydrogen bonds.26H30N2O4. Calculated, %: C, 71.89; H, 6.91; N, 6.45; O, 14.75.Elemental analysis : Found, %: C 71.87; H 6.91; N 6.45; O 14.74. C6.Uiso values of methyl H atoms were set to 1.5Ueq(C), while the Uiso values of H atoms bonded to the remaining C atoms were set to 1.2Ueq(C). The H atom bonded to O in the major occupied site was freely refined. The H atom bonded to O in the minor occupied site was refined using a riding model with Uiso(H) set to 1.5Ueq(O). In addition, the H—O—C—C torsion angle was allowed to refine. The displacement ellipsoids of O4 and O4′ were restrained to be similar. The distances O4—C3 and O4′—C3 were restrained to be similar. Bond lengths and angles in the fragments C24–O2–C27′ and C24–O2–C27 were restrained to be similar. The displacement ellipsoids of O2 and C27/C27′ were restrained to be similar. Bond lengths, angles and displacement parameters in the fragments N2–O3′–C31′–C32′–C33′–C34′–C35′–C36′–C37′–C8′ and N2–O3–C31–C32–C33–C34–C35–C36–C37–C8 were restrained to be similar. The following restraints implemented in SHELXL  were used to restrain the geometry  and ijU  of the disordered parts.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022006508/jq2018sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022006508/jq2018Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022006508/jq2018Isup3.cmlSupporting information file. DOI: 2092230CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II part of aquazinc(II) diperchlorate, [Zn(C8H20N4)H2O](ClO4)2, exhibits a slightly distorted square-pyramidal coordination environment with a water molecule in the apical position.The cationic Zn II part of aquazinc(II) bis(perchlorate), [Zn(C8H20N4)(H2O)](ClO4)2, exhibits a slightly distorted square-pyramidal coordination environment with a water molecule in the apical position. In the crystal, the macrocyclic ring alternates between two conformations with equal occupancies. Two of the three perchlorate anions are situated about a twofold rotation axis, and one of them shows disorder of the O atoms with occupancies of 0.62 (7) and 0.38 (7). In the crystal, the complexes are connected by intermolecular hydrogen bonding via the perchlorate anions.The cationic Zn The macrocyclic ring is disordered, and two alternate conformations of each N–C–C–N bridge can be observed (conformation A and B)  in the basal plane, with a ZnII-bound H2O molecule occupying the apical position. Addison et al. (1984β − α)/60°] to determine if the five-coordinate atom has a square-pyramidal or trigonal–pyramidal coordination environment. The bond angles β and α are the largest and second-largest in the coordination sphere, respectively; an ideal square pyramid and an ideal trigonal bipyramid have τ = 0 and 1, respectively. In conformation A, the N—ZnII—N bond angles α and β are 138.2 (3)° and 138.7 (3)°, respectively; the corresponding bond angles in conformation B are 137.4 (4)° and138.7(4)°. The τ values are 0.008 and 0.022 for conformations A and B, respectively. Therefore, the coordination geometry around the central ZnII cation can be described as slightly distorted square-pyramidal. The occupancies for the non-hydrogen atoms of cyclen except for the four carbon atoms  were set to 0.50. Atom Zn1 is 0.755 (5) and 0.763 (3) Å above the basal plane formed by four N atoms in conformations A and B, respectively. The Zn1—O1 bond length [1.9721 (4) Å] is within the typical range [1.94–2.03 Å] for similar five-coordinated Zn complexes 2  Fig. 1, in whic al. 1984 proposedII complex  I. DOI: 10.1107/S2414314621003977/vm4048Isup2.hklStructure factors: contains datablock(s) I. DOI: 2067247CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Three new styrylquinoline–chalcone hybrids are been syntheized using a three-step reaction sequence. In two of them, a combination of hydrogen bonds and π–π stacking interactions generates three-dimensional assemblies, but in the third, only a single weak π–π stacking interaction is present, linking the molecules into chains. 1H/13C NMR spectroscopy, and by high-resolution mass spectrometry, and the three products have been characterized by single-crystal X-ray diffraction. The molecular conformations of (E)-3-{4-[(E)-2-phenylethenyl]quinolin-2-yl}-1-(naphthalen-1-yl)prop-2-en-1-one, C30H21NO, (IVa), and (E)-3-{4-[(E)-2-(4-fluorophenyl)ethenyl]quinolin-2-yl}-1-(naphthalen-1-yl)prop-2-en-1-one, C30H20FNO, (IVb), are very similar. In each compound, the molecules are linked into a three-dimensional array by hydrogen bonds, of the C—H⋯O and C—H⋯N types in (IVa), and of the C—H⋯O and C—H⋯π types in (IVb), and by two independent π–π stacking interactions. By contrast, the conformation of the chalcone unit in (E)-3-{4-[(E)-2-(2-chlorophenyl)ethenyl]quinolin-2-yl}-1-(naphthalen-1-yl)prop-2-en-1-one, C30H20ClNO, (IVc), differs from those in (IVa) and (IVb). There are only weak hydrogen bonds in the structure of (IVc), but a single rather weak π–π stacking interaction links the molecules into chains. Comparisons are made with some related structures.Three new styrylquinoline–chalcone hybrids have been synthesized using a three-step pathway starting with Friedländer cyclocondensation between (2-aminophenyl)chalcones and acetone to give 2-methyl-4-styrylquinolines, followed by selective oxidation to the 2-formyl analogues, and finally Claisen–Schmidt condensation between the formyl intermediates and 1-acetylnaphthalene. All intermediates and the final products have been fully characterized by IR and  Compound (IIc): reaction time 14 h, yield 0.21 g (73%), yellow solid, m.p. 388–390 K, RF = 0.22 (12.5% ethyl acetate–hexane).Compound (IIa): reaction time 15 h, yield 0.19 g (86%), yellow solid, m.p. 367–369 K, v/v) to give the required formyl intermediates (IIIa)–(IIIc) as solid compounds.For the synthesis of compounds (III), a suspension of the appropriate 2-methyl-4-styrylquinoline (II) (1.0 mmol) and selenium dioxide (2.0 mmol) in 1,4-dioxane (5 ml) was stirred and heated at 373 K for the appropriate time. After the complete consumption of (II) [as monitored by thin-layer chromatography (TLC)], dichloromethane (15 ml) was added and the residual solid was removed by filtration. The solvent was removed under reduced pressure and the resulting crude products were purified by flash column chromatography on silica gel using hexane–ethyl acetate mixtures as eluent (compositions ranged from 7:1 to 2:1 RF = 0.31 (9.1% ethyl acetate–hexane). Compound (IIIb): reaction time, 1 h, yield 0.14 g (89%), yellow solid, m.p. 417–419 K, RF = 0.20 (9.1% ethyl acetate–hexane). Compound (IIIc): reaction time, 2 h, yield 0.21 g (92%), pale orange solid, m.p. 431–433 K, RF = 0.28 (9.1% ethyl acetate–hexane).Compound (IIIa): reaction time, 1 h, yield 0.23 g (96%), yellow solid, m.p. 421–423 K, For the synthesis of compounds (IV), a mixture of the appropriate 2-formyl intermediate (III) (1.0 mmol), 1-acetonaphthone (1.0 mmol) and potassium hydroxide (1.1 mmol) in ethanol (3 ml) was stirred at 298 K for the appropriate time. After complete consumption of (III) (monitored by TLC), the resulting precipitate was collected by filtration, washed with water (15 ml) and ethanol (10 ml), and then recrystallized from chloroform–ethanol to afford the target molecular hybrids (IV).RF = 0.22 (13% ethyl acetate–hexane). Compound (IVb)RF = 0.31 (13% ethyl acetate–hexane). Compound (IVc)RF = 0.20 (9% ethyl acetate–hexane).Compound (IVa)supporting information.Full details of the spectroscopic characterization are included in the Uiso(H) = 1.2Ueq(C).Crystal data, data collection and refinement details for compounds (IVa)–(IVc) are summarized in Table 1et al., 20221H/13C NMR spectroscopy, and by high-resolution mass spectrometry (HRMS); full details of the spectroscopic characterization are provided in the supporting information.We have recently reported  and (IIc) matched perfectly those of previously reported analogues –(IVc). The formation of molecular hybrids (IV) was established by disappearance of the formyl signals from both the 1H and 13C NMR spectra, and by the appearance of signals from the newly formed 3-arylpropen-1-one fragment. As far as the Claisen–Schmidt condensation is concerned, it proceeded in a highly stereoselective manner, giving exclusively the E-stereoisomers, as indicated by the 1H NMR spectra. The trans configuration of the arylpropen-1-one fragment was deduced on the basis of the coupling constant values  between HA and HB , whose signals in the 1H NMR spectra appear at δ 7.91–7.93 and 7.78–7.79, respectively.The presence of stretching vibration bands in the range 1727–1731 cmE-configuration of both the styryl and the chalcone moieties –(IVc) which fully confirm the molecular structures deduced from the spectroscopic data, in particular, the s Figs. 1–3 ▸ ▸. et al., 2022et al., 2022ca 180° around the C22—C23 bond (Table 2For each of (IVa)–(IVc), the atom labelling Figs. 1–3 ▸ ▸ fet al., 1998abet al., 2000et al., 1990et al., 1995The supramolecular assembly in compound (IVa)n, n) alternate with n, n), where n represents an integer in each case. The pyridine rings of the molecules at  and  are strictly parallel with an interplanar spacing of 3.2877 (5) Å and a ring-centroid separation of 3.5372 (7) Å, corresponding to a ring-centroid offset of 1.305 (2) Å. This interaction links the x, y, z) and the styryl ring at  make an interplanar angle of only 6.37 (7)°; the ring-centroid separation is 3.7818 (9) Å and the shortest perpendicular distance between the centroid of one ring and the plane of the other is 3.4535 (6) Å, corresponding to a ring-centroid offset of 1.541 (2) Å. This interaction links the The linking of these dimeric units by C—H⋯N hydrogen bonds gives rise to a ribbon running parallel to the  and quinoline, the molecules are linked into cyclic centrosymmetric dimers by hydrogen bonds, of the C—H⋯N and C—H⋯π types, respectively, and these dimers are further linked by π–π stacking interactions to form sheets in the fluoro compound and chains in the trifluoromethyl analogue. By contrast, there are no significant intermolecular interactions in the structure of (E)-4--2-methylquinoline. All of these type (II) compounds have molecular skeletons in which the styryl and quinoline units are non-coplanar, as reported here for compounds (IVa)–(IVc). This appears to be the case for all of the 4-styrylquinolines which have been structurally characterized so far, in contrast to the 2- and 8-styrylquinolines, where the two ring systems appear always to be effectively coplanar  three products and all of the intermediates on the pathways leading to them, and we have determined the molecular and supramolecular structures of the three products.We have developed a highly versatile and efficient three-step synthesis of a novel class of styrylquinoline–chalcone hybrids based on only very simple and readily available starting materials, such as simple aldehydes and ketones, and we have characterized by spectroscopic means  global, IVa, IVb, IVc. DOI: 10.1107/S2053229622011263/ov3164IVasup2.hklStructure factors: contains datablock(s) IVa. DOI: 10.1107/S2053229622011263/ov3164IVbsup3.hklStructure factors: contains datablock(s) IVb. DOI: 10.1107/S2053229622011263/ov3164IVcsup4.hklStructure factors: contains datablock(s) IVc. DOI: 10.1107/S2053229622011263/ov3164sup5.txtSpectroscopic data. DOI: 2221749, 2221750, 2221751CCDC references:


A and B) are present in the asymmetric unit, with different conformations. The dihedral angle between the mean planes of the carbazole systems for molecule A is 49.1 (2)° compared to 84.0 (1)° for molecule B. In the crystal, numerous aromatic π–π stacking interactions [shortest centroid–centroid separation = 3.7069 (19) Å] help to establish the three-dimensional supramolecular network.In the title compound, two independent molecules  are present in the asymmetric unit, with different conformations. The dihedral angle between the mean planes of the carbazole systems for molecule A is 49.1 (2)° compared to 84.0 (1)° for molecule B. In the crystal, numerous aromatic π–π stacking interactions [shortest centroid–centroid separation = 3.7069 (19) Å] help to establish the three-dimensional supramolecular network.In the title compound, C Two independent molecules (A and B) are present in the asymmetric unit, with different conformations. Each of the independent molecules is composed of two carbazole systems connected by an iodobenzene bridge ° compared to 84.0 (1)° for the N3 and N4 ring systems in molecule B. In the crystal, numerous face-to-face [shortest centroid–centroid separation = 3.7069 (19) Å] and edge-to-face π–π stacking interactions connect the molecules into a three-dimensional supramolecular network , t-BuOK , and dimethyl sulfoxide (10 ml) was stirred at 120°C before 1-iodo-3,5-difluorobenzene  was injected. The reaction mixture was stirred at 140°C for 2 h. After being cooled, the mixture was extracted with chloroform and the organic extracts were combined, washed with water, and the organic layer was dried over anhydrous MgSO4. After evaporating the solvent, the crude product was purified by column chromatography on silica gel with chloroform/n-hexane as the eluent to give a white powder. Yield: 83%. 1H NMR  δ 8.17 , 8.08 , 7.84 , 7.56 , 7.49 , 7.35 . Colourless blocks were obtained by recrystallization from mixed solvents of methylene chloride and n-hexane (v:v = 1.2).A mixture of 9Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2414314621004284/hb4376sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314621004284/hb4376Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621004284/hb4376Isup3.cmlSupporting information file. DOI: 2079137CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Nickel(II) Schiff base complexes containing thiolate S and polyamine N donor atoms exhibit electrocatalytic activity for proton reduction. The piperazine moiety in the Schiff base ligand gives a smaller bite angle, which is effective in reducing the overpotential. N)(methyl)amino-κN]ethyl}imino-κN)methyl]benzenethiolato-κS}nickel(II) chloride, [Ni(C12H18N3S)]Cl (1), and [2-ethyl]imino-κN}methyl)benzenethiolato-κS]nickel(II) hexafluorophosphate dichloromethane monosolvate, [Ni(C13H18N3S)]PF6·CH2Cl2 (2), were synthesized by the reactions of 2-(tert-butylthio)benzaldehyde, triamines, and nickel(II) salts. Both complexes have a nickel ion surrounded by an N,N′,N′′,S-tetradentate ligand, forming a square-planar geometry. The terminal N,N-chelating moiety is N,N-dialkylethane-1,2-diamine for 1 and 1-alkylpiperazine for 2. The N—Ni—N bite angle in the terminal N,N-chelate ring in 2 [76.05 (10)°] is much smaller than that in 1 [86.16 (6)°]. Cyclic voltammograms of 1 and 2 in aqueous media indicated that the reduction and oxidation potentials of 2 are more positive than those of 1. The smaller bite angle of the terminal piperazine chelate in 2 reduces the electron-donating ability of the tetradentate ligand, resulting in a positive shift of the redox potentials. Both complexes exhibit catalytic activity for proton reduction, and the piperazine moiety in 2 is effective in reducing the overpotential.The thiolate nickel complexes {2-[({2-[(2-aminoethyl-κ In the electrocatalytic proton reduction, the electrochemical properties of the precatalysts are directly related to the formation of real catalysts. Therefore the tuning of the redox properties is also required in the ligand design.The nickel(II) complex Cl (1) and [Ni(C13H18N3S)]PF6 (2), in which the N,N′,N′′,S-tetradentate Schiff base ligands contain an additional N-methyl group and a terminal piperazine moiety, respectively. The electrochemical properties of these complexes were investigated by cyclic voltammetry in water, and compared with those of [Ni(C11H16N3S)]Cl (3) without N-substituents.In this work we synthesized two water-soluble N2.1 and 2 consist of an Ni2+ ion and a monoanionic N,N′,N′′,S-tetradentate ligand, giving a square-planar geometry. The asymmetric unit in 1 comprises the complex cation and a chloride anion, whereas in 2 a hexafluorophosphate anion and a dichloromethane molecule are incorporated into the crystal lattice.The complex cations in N,S-chelate, the N1—Ni1—S1 angles are 97.77 (5)° in 1 (Table 12 (Table 23 [98.5 (2)°] and the tetraphenylborate salt [Ni(C11H16N3S)]B(C6H5)4 . The bond distances in the chelate rings are also comparable ° in 1, 87.80 (10)° in 2, 86.1 (3)° in 3  complexes with S,N,N,S-tetradentate ligands, which have two amine N or two imine N donor atoms besides two S atoms °] is much smaller than those of 1 [86.16 (6)°], 3  and 2 [N1—C8—C9—N2 = 45.1 (3)°], this bending is due to the rigid structure of the piperazine chelate, which fixes the direction of the methylene groups on the tertiary amine N atom.In complex 3.1 shows hydrogen bonds between the terminal amine nitrogen atom in the complex cation and two chloride ions with the N3⋯Cl1 and N3⋯Cl1 and 3.8965 (13) Å].The crystal structure of y Table 3, which aon Fig. 3. The dis2 between the methylene hydrogen atoms of the polyamine moiety and the π system of the benzene ring  distance of 3.114 (3) Å  = 2.47 (4) Å].Several intermolecular C—H⋯π interactions exist in g Table 4. The pipÅ Table 4. In addi4.N,N′,N′′,S-tetradentate Schiff base ligands studied here have not been reported so far for other transition-metal ions. A similar Schiff base structure that contains benzenethiolate and polyamines is found in a trinuclear nickel(II) complex with a C3-symmetric ligand based on a 1,3,5-trimercaptobenzene backbone °  complexes 1, 2, and 3 was investigated by cyclic voltammetry. Measurements were performed in 5 × 10−4M (1 M = 1 mol dm−3) aqueous solution containing KNO3 (0.1 M) at a scan rate of 0.1 V s−1. The working electrode was a glassy carbon disk electrode with a diameter of 3 mm, the auxiliary electrode was a platinum wire, and the reference electrode was Ag/AgCl/saturated KCl. All complexes exhibit irreversible reduction and oxidation processes  suggest the adsorption of the reduced species. In the oxidation process, the anodic wave appeared at 0.73 V for 1, 0.79 V for 2, and 0.68 V for 3. In both processes, the redox potentials of 1 are slightly shifted to more positive values than those of 3, which suggests that the electronic and steric effects of the methyl group on the central N atom are not so significant. The voltammogram of 2 shows further positive shifts, and the shift in the reduction process is more pronounced. This is probably related to the smaller bite angle of the terminal piperazine chelate, which reduces the electron-donating ability of the Schiff base ligand toward the nickel center.The redox behavior of the es Fig. 5. In the 1 and 2 were compared in a buffer solution of pH 4.6 (0.1 M acetic acid/sodium acetate). A catalytic current was observed during the reduction process, giving a peak at −1.28 V for 1 and −1.23 V for 2  under N2. Elemental analyses were performed by the Analytical Research Service Center at Osaka City University or A Rabbit Science Co., Ltd.12H18N3S)]Cl (1).PF6 (2).[Ni(C To a solution of N-(2-aminoethyl)piperazine  and 2-(t-butylthio)benzaldehyde  in ethanol (20 mL) was added NiCl2·6H2O . The resulting suspension was refluxed under a nitrogen atmosphere for 8 h, during which time the color of the solution turned orange and a yellow–green precipitate formed. The reaction mixture was filtered, and an ethanol solution (5 mL) of ammonium hexafluorophosphate  was added to the filtrate. The resulting orange precipitate was collected by filtration and dried under reduced pressure to give an orange powder of 2 . Suitable crystals for X-ray diffraction analysis were grown from a dichloromethane solution by layering with diethyl ether. 1H NMR : δ 2.76–2.94 2NH), 2.83 , 3.91–4.03 2NH), 4.16–4.27 2NH), 4.31 , 7.10 , 7.28 , 7.52 , 7.74 , 8.16 . Analysis calculated for C13H18F6N3NiPS·0.75CH2Cl2: C, 32.02; H, 3.81; N, 8.15. Found: C, 32.11; H, 3.93; N, 8.14.8.N,N-chelating moieties in 1 were modeled as disordered over two positions each, and the occupancy factors refined to 0.864 (3) and 0.136 (3). Hydrogen atoms on the disordered C atoms and the adjacent C atoms that belong to the minor site were placed in calculated positions with C—H(methyl) = 0.98 Å and C—H(methylene) = 0.99 Å and refined using a riding model with Uiso(H) = 1.5Ueq(C) and 1.2Ueq(C), respectively. Other H atoms were found in a difference-Fourier map and freely refined.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989022003954/dj2045sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989022003954/dj20451sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989022003954/dj20452sup3.hklStructure factors: contains datablock(s) 2. DOI: 2165891, 2165890CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II ions both show a distorted N3Cl3 octahedral coordination environment defined by three N atoms of the tridentate 2,4,6-tri-2-pyridyl-1,3,5-triazine ligand, two bridging Cl− ligands and a Cl− anion.The Ni 2Cl4(C18H12N6)2], the NiII ions are hexa-coordinated in a distorted octahedral coordination environment defined by three N atoms of the tridentate 2,4,6-tri-2-pyridyl-1,3,5-triazine ligand and three Cl− anions in a meridional geometry. The two NiII ions are bridged by two Cl anionic ligands, thereby forming a dinuclear complex. A crystallographic centre of inversion is located at the centroid of the Ni2Cl2 ring.In the title compound, [Ni On the other hand the Cl2—Ni1—Cl1i axis (symmetry code: (i) −x, −y + 1, −z) is almost linear [Cl2—Ni1—Cl1i = 176.25 (2)°]. The Ni—N(pyridyl) bonds [Ni1—N4/N6 = 2.130 (2) and 2.129 (2) Å] are slightly longer than the Ni—N(triazine) bond [Ni1—N1 = 1.970 (2) Å]. The three Ni—Cl bond lengths are somewhat different . The two pyridyl rings that coordinat to the NiII atom are located approximately parallel to the respective triazine ring, making dihedral angles of 4.51 (6) and 4.95 (6)°, respectively. The dihedral angle between the non-coordinating pyridyl substituent and the triazine ring is 7.56 (6)°.In the complex, the two Niex Fig. 1. Each NiCg1 (the centroid of ring N5/C8–C12) and Cg2ii [the centroid of ring N6/C14–C18; symmetry code: (ii) x, −y + z − The complex displays numerous intermolecular π–π interactions between adjacent six-membered rings. For e Table 1.2·6 H2O  in ethanol (30 ml) was added 2,4,6-tri-2-pyridyl-1,3,5-triazine . The solution was stirred for 12 h at room temperature. The formed precipitate was separated by filtration, washed with ethanol and acetone, and dried at 323 K, to give a pale-green powder . Brown crystals suitable for X-ray analysis were obtained by slow evaporation from a dimethyl sulfoxide (DMSO) solution at 363 K.To a solution of NiCl−3) and minimum (−0.20 e Å−3) electron density in the difference Fourier map are located 0.73 and 1.28 Å, respectively, from atoms C2 and C9.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621000936/im4011sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314621000936/im4011Isup2.hklStructure factors: contains datablock(s) I. DOI: 2058987CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The units are further linked by O—H⋯O and N—H⋯Cl hydrogen bonds.In the crystal of the title organic salt, the cation and the anion are connected  H-benzimidazol-3-ium] 2,5-dichloro-3,6-dioxocyclohexa-1,4-diene-1,4-diolate}, 2C13H11N2O+·C6Cl2O42−, the chloranilate anion is located on an inversion centre, so that the asymmetric unit contains one cation and one half of the chloranilate anion. In the crystal, the cation and the anion are connected by a bifurcated N—H⋯ hydrogen bond, forming a 2:1 unit. The units are linked into a layer lying parallel to -1 D—H⋯A hydrogen bonding  in chloranilic acid–organic base systems ; symmetry code as given in Table 1via O—H⋯O and N—H⋯Cl hydrogen bonds  Å and C2—Cl1⋯Cg3iv = 139.21 (5)°; symmetry code: (iv) x, y, z − 1] is observed between the layers, where Cg3 is the centroid of the C11–C16 ring.We have prepared the title compound in order to continue our studies of H-benzimidazole in a ca 1:1 molar ratio at room temperature [150 ml methanol solution of chloranilic acid (0.45 g) and 2-(2-hydroxyphenyl)-1H-benzimidazole (0.45 g)].Single crystals of the title salt were obtained by slow evaporation from a methanol solution of chloranilic acid with 2-(2-hydroxyphenyl)-1Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314621011500/hb4396sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314621011500/hb4396Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621011500/hb4396Isup3.cmlSupporting information file. DOI: 2119367CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Targeted drug delivery systems hold the remarkable potential to improve the therapeutic index of anticancer medications markedly. Here, we report a targeted delivery platform for cancer treatment using clathrin light chain (CLC)‐conjugated drugs. We conjugated CLC to paclitaxel (PTX) through a glutaric anhydride at high efficiency. Labeled CLCs localized to 4T1 tumors implanted in mice, and conjugation of PTX to CLC enhanced its delivery to these tumors. Treatment of three different mouse models of cancer—melanoma, breast cancer, and lung cancer—with CLC‐PTX resulted in significant growth inhibition of both the primary tumor and metastatic lesions, as compared to treatment with free PTX. CLC‐PTX treatment caused a marked increase in apoptosis of tumor cells and reduction of tumor angiogenesis. Our data suggested HSP70 as a binding partner for CLC. Our study demonstrates that CLC‐based drug‐conjugates constitute a novel drug delivery platform that can augment the effects of chemotherapeutics in treating a variety of cancers. Moreover, conjugation of therapeutics with CLC may be used as means by which drugs are delivered specifically to primary tumors and metastatic lesions, thereby prolonging the survival of cancer patients. ACNacetonitrileADCantibody‐drug conjugateBUNblood urea nitrogenCD31cluster of differentiation 31CLCclathrin light chainDAPIdiamidino‐2‐phenylindoleDLSdynamic light scatteringDMEMDulbecco's modified Eagle's mediumDMSOdimethyl sulfoxideECMextracellular matrixEDC1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimideESIMSelectron spay ionization mass spectrometryE. coliEscherichia coliFBSfetal bovine serumGAPDHglyceraldehyde‐3‐phosphate dehydrogenaseHPLChigh‐performance liquid chromatographyHRPhorseradish peroxidaseHSP70heat shock protein 70H&Ehematoxylin and eosinLLC1Lewis lung carcinoma 1LYVE1lymphatic vessel endothelial hyaluronan receptor 1MALDI‐TOFmatrix‐assisted laser desorption ionization time‐of‐flightMFImean fluorescence intensityMSmass spectrometryNHSN‐hydroxysuccinimideNMRnuclear magnetic resonancePDBprotein data bankPKpharmacokineticsPTXpaclitaxelROIregion of interestRPMIRoswell park memorial instituteSDstandard deviationSDS‐PAGEsodium dodecyl sulfate‐polyacrylamide gel electrophoresissiRNAsmall interfering ribonucleic acidTDLNtumor‐draining lymph nodeTGItumor growth inhibitionUV–Visultraviolet–visible1Targeted drug delivery systems can amplify the concentration of transported payloads at various tissues of interest, including tumors.Antibody‐drug conjugates (ADCs) are emerging platforms for the delivery of a range of cancer drugs.Here, we report a novel application of clathrin light chain A (CLC)‐based drug delivery. CLC is an endogenous small protein that forms a network of triskelions that constitute a polyhedral lattice around vesicles that assist in the sorting of cargo for intracellular trafficking.First, we confirmed that CLCs interact with cancer cells and could be used as vehicles for targeted delivery to malignant tumors. Next, CLCs were conjugated to the antineoplastic agent paclitaxel (PTX) via reaction with glutaric anhydride (CLC‐PTX) to evaluate their utility for cancer‐targeted delivery and treatment in a series of mouse models. CLC‐PTX enhanced the concentration of PTX at the tumor site and suppressed tumor growth in mouse models of breast cancer, melanoma, and lung carcinoma. In addition, CLC‐PTX was found to reduce the size of metastatic lesions in breast cancer.22.1Escherichia coli (E. coli), confirmed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and extracted at a purity of >85%, as demonstrated by Western blot (data not shown). PTX is a chemotherapeutic drug used for the treatment of numerous cancers.1H‐NMR  was expressed in R Figure  and 13C‐R Figure . In the N‐hydroxysuccinimide (NHS) coupling to produce CLC‐PTX*, which was verified by ultraviolet–visible (UV–Vis) spectroscopy  was used to confirm the conjugation of PTX to CLC. The maximal absorbance of PTX* is observed at a wavelength of around 500 nm. We conjugated 2′‐glutaryl PTX* to CLC via 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC)/sulfo‐y Figure . The absy Figure . After cy Figure , whereasy Figure . Next, Cy Figure . Intravey Figure A. In addy Figure B and C.2.2We sought to determine whether CLC was internalized by 4T1 mouse breast cancer cells by incubating these cells with CLCs attached to Alexa Fluor™ 488 dye. The colocalization of the CLCs with a lysosome marker was confirmed Figure , which iA virtual screening between CLC and a protein data bank (PDB) was conducted, resulting in the identification of three proteins with high binding energy Table . Interes2.3iv to 4T1 tumor‐bearing BALB/c mice. The mice were euthanized at 1 day (1 d), 2 days, and 3 days following administration of CLCs, at which time points we measured the fluorescent signal of CLC‐IR800 in various organs, using an iBox Exploreriv with CLC‐PTX* and quantified the fluorescence of PTX*. PTX* did not clear significantly from the tumor within the span of the study, whereas the PTX* signal declined in the liver and the kidney over time  e Figure .The lung is the most common site of metastasis for breast cancer. Lung with 4T1 metastasis showed enhanced targeting signals of CLC as compared with naïve lung Figure . Interesiv administration to 4T1 breast tumor‐bearing BALB/c mice. Ex vivo fluorescent images of the tumors were acquired 1 day following administration of PTX* or CLC‐PTX* at an equivalent PTX* dose of 0.5 mg/kg, which showed that the accumulation of CLC‐PTX* in the tumor was higher than free PTX*  quantified PTX. We detected significant amounts of PTX in the sera of the CLC‐PTX group as compared with the free PTX group at 6 h, indicating that conjugation to CLC enhanced the blood circulation of PTX . The tumors in the CLC‐PTX group displayed slower tumor progression in comparison to the two other treatment groups  of 4T1 tumor‐bearing mice at 27 days following implantation Figure . The exp2.63, following a schedule and dosing identical to the 4T1 study above. Again, CLC‐PTX displayed the most potent inhibitory effect on tumor growth: at the end of the study, the tumors of the mice treated iv with CLC‐PTX were the smallest (0.81 ± 0.30 × 103 mm3), as compared to the PBS‐treated mice (2.36 ± 0.61 × 103 mm3) and free PTX‐treated mice (1.29 ± 0.50 × 103 mm3) . Immunostaining demonstrated a decline of Melan‐A+ cells with an increase in caspase‐3 expression in the CLC‐PTX‐treated mice as compared to other groups  Figure . TGI by s Figure . The B16s Figure .2.7iv to C57BL/6 mice bearing LLC1 murine lung carcinoma, following a schedule identical and dosing in the aforementioned tumor models. The tumors in the CLC‐PTX group (0.80 ± 0.21 × 103 mm3) were significantly smaller than those in the mice that received PBS (1.68 ± 0.30 × 103 mm3) and free PTX (1.21 ± 0.31 × 103 mm3) . Next, we stained LLC1 tumor tissue sections with the cancer marker pan‐cytokeratin, apoptosis marker caspase‐3, proliferation marker Ki67, and vascular marker CD31, and examined them by fluorescence microscopy. Treatment with CLC‐PTX reduced the amount of Pan‐Cytokeratin+ cancer cells significantly, whereas Caspase‐3+ apoptotic cells increased  Figure . In addid Figure . Consistd Figure . Furtherd Figure . Thus, t3Targeted drug delivery systems have attracted major interest and already entered into clinical practice for various diseases, but their application to cancer remains to be developed.Metastasis is the primary factor of cancer morbidity and mortality.Nonetheless, future in vivo targeting studies are required to define the mechanisms by which HSP70 functions as a target of CLC‐conjugated drug delivery. In addition, HSP70 produced by tumor cells may undergo differential glycosylation, so kinetic studies tailored to each type of cancer may be required for more specific and accurate assessment of the binding affinity of HSP70 to CLC. The performance of a mutational study to identify putative binding site of CLCOur data suggest that CLC targeted the primary tumor and metastatic lung lesions of 4T1 breast cancer in mice with high efficacy. While there was an increase in CLCs in the peripheral organs, in particular, the kidney; however, this retention faded after 3 days. Nonetheless, the tumor uptake of exogenously administered CLCs remains relatively stable over time.CLC‐PTX enhanced the therapeutic efficacy of PTX on the primary 4T1 tumor, as demonstrated by suppression of tumor growth, enhancement of cancer cell apoptosis, and especially inhibition of angiogenesis. CLC‐PTX treatment was also found to reduce the density of lymphatic vasculature, which often contributes to the immunosuppressive environment of TDLN. Concomitantly, distant lung metastasis was also inhibited more effectively by treatment with CLC‐PTX than free PTX. Excessive accumulation of extracellular matrix (ECM) in the tumor stroma is referred to as the desmoplastic reaction, commonly reported as a major obstacle to the treatment of cancers.44.14T1 mouse breast cancer (ATCC® CRL‐2539™), B16 mouse melanoma (ATCC® CRL‐6322™), LLC1 mouse lung carcinoma (ATCC® CRL‐1642™), and HK02 human kidney tubular epithelial cell lines (ATCC® CRL‐2190™) were purchased from American Type Culture Collection . 4T1, B16, LLC1, and HK02 cells were cultured in RPMI‐1640 or Dulbeccos modified Eagles medium with 10% fetal bovine serum and 1% penicillin/streptomycin (pen/strep).4.2C57BL/6 (JAX# 000664) and BALB/c (JAX# 000651) mice were obtained from The Jackson Laboratories. All animal experiments and methods were performed in accordance with the relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee (protocols: 2016 N000167/04977) of Brigham and Women's Hospital .4.3E. coli expression system as follows. The human CLC gene was codon‐optimized for expression in E. coli and chemically synthesized by Biomatik. The synthesized gene was subsequently cloned into NdeI/HindIII sites with pET30a vector. These expression plasmids were used to transform the E. coli BL21(DE3). His‐tag was applied to a Ni‐NTA column and eluted in a buffer containing 10 mM Tris, 0.15 M NaCl, 8 M urea, and 0.3 M imidazole at a pH of 8.0.Human CLC was expressed and optimized for an Gene sequence; ATGGCGGAACTGGACCCGTTCGGCGCTCCGGCAGGCGCACCGGGCGGTCCGGCGCTGGGTAACGGCGTTGCGGGTGCTGGTGAAGAAGACCCGGCAGCAGCGTTCCTGGCGCAGCAGGAATCTGAAATCGCAGGTATCGAAAACGATGAAGCGTTCGCGATCCTGGACGGTGGTGCTCCGGGTCCGCAGCCGCACGGTGAACCGCCGGGTGGTCCGGATGCGGTTGACGGTGTTATGAACGGCGAGTACTACCAGGAGTCTAACGGTCCGACCGATTCTTACGCGGCAATTAGCCAGGTTGATCGTCTGCAATCCGAACCGGAATCTATCCGTAAATGGCGTGAGGAGCAGATGGAACGCCTGGAAGCTCTGGACGCGAACTCTCGCAAACAGGAGGCGGAATGGAAAGAAAAAGCGATCAAAGAGCTGGAAGAATGGTATGCGCGTCAGGACGAACAGCTGCAAAAAACCAAAGCGAACAACCGTGTGGCGGACGAAGCATTCTACAAACAGCCGTTTGCGGACGTTATCGGTTACGTTACCAACATCAACCATCCGTGCTACTCTCTGGAGCAGGCAGCGGAAGAAGCGTTCGTGAACGACATCGACGAATCTAGCCCAGGCACCGAATGGGAACGTGTTGCGCGCCTGTGCGACTTCAACCCGAAATCTTCTAAACAGGCTAAAGACGTTTCTCGTATGCGTTCTGTTCTGATCTCTCTGAAGCAGGCTCCGCTGGTTCAC.Amino sequence; MAELDPFGAPAGAPGGPALGNGVAGAGEEDPAAAFLAQQESEIAGIENDEAFAILDGGAPGPQPHGEPPGGPDAVDGVMNGEYYQESNGPTDSYAAISQVDRLQSEPESIRKWREEQMERLEALDANSRKQEAEWKEKAIKELEEWYARQDEQLQKTKANNRVADEAFYKQPFADVIYVTNINHPCYSLEQAAEEAFVNDIDESSPGTEWERVARLCDFNPKSSKQAKDVSRMRSVLISLKQAPLVH.The purity of CLC was >85%, as estimated by a Coomassie blue‐stained SDS‐PAGE gel. The concentration of CLC was determined by Bradford protein assay, using BSA as a standard. Its molecular weight was 28.1 kDa, and its isoelectric point was 4.37.4.4www.rcsb.org. Three proteins with the highest binding energy were identified through AutoDock Vina: ADP‐ribosylation factor 3, HSP70, and serine/threonine‐protein phosphatase PP1‐beta catalytic subunit  was labeled with Alexa Fluor™ 488 NHS Ester (Thermo Fisher Scientific) or Alexa Fluor™ 594 NHS Ester (Thermo Fisher Scientific) and incubated with 4T1 cells for 2 h at 37°C. After five washes with PBS buffer, 4T1 cells were stained with lysosomal staining kit . 4T1 cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), and diamidino‐2‐phenylindole (DAPI)  was used to counterstain the cell nuclei. The cells were visualized using an EVOS™ FL Auto 2 Imaging System (Thermo Fisher Scientific). After splitting each color channel, the area with fluorescence was measured for each color channel with Image J . The percent of uptake of CLC was calculated by dividing with measured area from DAPI channel.4.62O with 0.1% formic acid for 20 min), using a Phenomenex Luna 5 μm C18 column . The product was detected at 14.1 min, and molecular weight was confirmed as 969 g/mol by electron spay ionization mass spectrometry analysis ([M‐H]− m/z at 968]). 2'‐Glutaryl PTX was purified by reversed‐phase HPLC  with a gradient solvent system (15%–75% ACN/H2O with 0.1% formic acid for 40 min). The product was eluted at a retention time of 13.4 min under these HPLC conditions. 2′‐Glutaryl PTX was confirmed by 1H‐NMR (proton nuclear magnetic resonance) and 13C‐NMR (carbon nuclear magnetic resonance) spectra. 2'‐glutaryl PTX (0.2 mg) dissolved in dimethyl sulfoxide (DMSO) (Thermo Scientific Fisher) was activated with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride  and Sulfo‐NHS (N‐hydroxysulfosuccinimide)  for 15 min at RT in 2‐(N‐morpholino)ethanesulfonic acid (MES) buffer  . The EDC was quenched by 2‐mercaptoethanol  for 10 min. Immediately, the pH of the solution was increased by NaHCO3  to ~8. CLC dissolved in PBS  was mixed with the activated 2′‐glutaryl PTX for 2 h at RT . Dialysis was performed twice by a centrifugal filter  at 10,000 rpm for 15 min to remove the free PTX. The solution was purified further by a desalting column .Glutaric anhydride  and PTX  were prepared in a 4 ml vial dried under high vacuum for 24 h and dissolved in 1 ml of pyridine. The solution was stirred at room temperature under Ar atmosphere for 2 h. The mixture was diluted with 300 μl of methanol, and 5 μl of solution was injected into liquid chromatography/mass spectrometery (LC/MS)  with a gradient reversed‐phase system (10%–100% ACN/H4.7280 nm) of CLC was extracted from the absorption spectrum of CLC solutions at different concentrations. The εCLC,280 nm was 4.8 × 104, whereas εPTX,500 nm was 4.2 × 104. The concentration of CLC (CCLC) in the CLC‐PTX* solution was calculated by /εCLC,280 nm, and CPTX* was by .Absorbance was measured by UV–Vis spectroscopy . The molar extinction coefficient  at 1:1 ratio. The 2 μl of the samples was spotted, air‐dried, and detected with a linear detector. The data were analyzed using Bruker Daltonics flexAnalysis. External calibration was performed with BSA with [M‐H] average mass (m/z) of 66,428.MALDI mass analyses of CLC and CLC‐PTX were performed with a Bruker MicroFlex™ MALDI‐TOF in negative mode. The protein solution was mixed with sinapinic acid matrix solution  was incubated in acetate buffer  or phosphate buffer  at 37°C. The solutions were dialyzed with 5% DMSO solution, and filtrates were analyzed by UV–Vis spectroscopy to quantify the amount of free PTX* in comparison to the absorbance of the solution prior to incubation.4.105) was stained with CLC‐Alexa 594 or anti‐HSP70‐Alexa 594 in FACS buffer  at 4°C for 30 min. Cells were washed by FACS buffer and fixed with FACS buffer containing 1% formalin. The autofluorescence from the cell itself was unmixed and only single cells were gated (Cytek® Aurora). Analysis of flow cytometry data was performed by FlowJo software . The histogram over Alexa 594 channel was extracted and the total fluorescence signal was divided by the total number of the cell.The surface of 4T1 cells (1.0 × 104.11Cells were transfected with Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Briefly, cells were plated at 20%–30% density in 12‐well plates 24 h prior to transfection. For siRNA transfection, the equivalent of 200 nM of siRNA per well of a 12‐well plate was utilized. After a 48 h incubation period, the lysates of the cells were measured using the Bradford assay. Equal amounts of protein were separated by SDS‐PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were immunoblotted with the following specific antibodies: anti‐HSP70, anti‐rabbit IgG‐HRP (Sigma‐Aldrich), anti‐rat IgG‐HRP (Sigma‐Aldrich), and anti‐GAPDH (Sigma‐Aldrich), using standard protocols. The blots were developed with West Dura chemiluminescent substrates using a Bio‐Rad ChemiDoc imaging system.4.12s.c. with 1.0 × 105 4T1 cells. At 14 days postimplantation, these mice received a single iv injection of CLC‐IR800 conjugate (4 mg/kg). At 1 day, 2 days, and 3 days following injection, the mice were euthanized via carbon dioxide inhalation and cervical dislocation, and their major organs were harvested and imaged by using a UVP iBOX Explorer Imaging Microscope (UVP) equipped with a 750–780 nm band‐pass excitation filter and an 800 nm long‐pass emission filter. MFI for each organ was measured using the region of interest (ROI) function of ImageJ .Animal studies were approved and conducted according to the Institutional Animal Care and Use Committee of Brigham and Women's Hospital, Boston, MA. CLC‐IR800 was prepared by incubating CLC with IRdye 800CW NHS ester  for 2 h at RT. The crude was purified by a desalting column . For ex vivo biodistribution studies, BALB/c mice were injected 4.13s.c. implanted with 1.0 × 105 4T1 cells. At 14 days postimplantation, these mice received a single iv injection of CLC‐PTX* conjugate . After 1 day, the mice were euthanized via carbon dioxide inhalation and cervical dislocation, and their major organs were harvested and imaged by using a UVP iBOX Explorer Imaging Microscope (UVP) equipped with a 455–495 nm band‐pass excitation filter and a 513–557 nm band‐pass emission filter. MFI of the organs was measured following subtraction of autofluorescence, using the ROI function of ImageJ.CLC‐PTX* was prepared with the identical protocol for the synthesis of CLC‐PTX except the use of PTX* . BALB/c mice were 4.1418: 100 × 2.1 mm, 2.6 μm) under the following gradient elution system: 10% acetonitrile/water to 100% acetonitrile/water for 20 min with a flow rate of 0.3 ml/min. Extracted ion is 854 m/z.BALB/c mice  were injected intravenously with PTX or CLC‐PTX at an equivalent PTX dose of 2.5 mg/kg. Sera from three mice were collected at 6 h and 2 days following drug administration. The serum samples were stored at −20°C until analysis. Serum samples (200 μl) were mixed with acetate buffer  for 1 day at 37°C. PTX in the serum was extracted by incubation with acetonitrile (6 ml) for 2 h at 37°C. After centrifugation , the supernatants were analyzed by HPLC. High‐resolution electrospray ionization mass spectra were acquired using Agilent LC‐q‐TOF Mass Spectrometer 6530‐equipped with a 1290 uHPLC system. The collected samples were injected into a reversed‐phase HPLC column  were 4.165 4T1 cells in the left fourth mammary gland. C57BL/6 mice  were inoculated subcutaneously with 1.0 × 105 B16, LLC1, or Pan02 cells in the right rear flanks. When the tumor size reached ~100 mm3, the mice were randomly divided into three groups (n = 6). All groups received treatments iv; the first group was injected with PBS (control), the second group with free PTX , and the final group with CLC‐PTX . The treatment schedule consisted of twice‐per‐week injections for 2 weeks. The tumor size and body weight of the mice were monitored during the treatment course. The length (l) and width (w) of the tumor was measured by a digital Vernier caliper, and tumor volume (V) was defined as V=l×w2/2. We evaluated the TGI rate of each group (TGI%=Vc−Vt/Vc−Vi×100), in which Vc is the volume of the control tumor at the end of the study, Vt is the volume of drug‐treated tumor at the end of the study, and Vi is the volume of tumor at the initial treatment.BALB/c mice  were implanted subcutaneously with 1.0 × 10Before the diameter of the tumor reached ~2 cm, the mice were euthanized, and lungs, livers, kidneys, spleen, tumor, and draining inguinal lymph nodes were harvested and embedded in optimum cutting temperature (OCT) compound .4.17Frozen OCT blocks of tumors and LNs were cut using a cryostat (Leica) into 8‐μm thick sections and cells were incubated in glass‐bottomed well plate (Lab Tek™). Meanwhile, cell samples were also stained by the following procedure. The samples were stained using anti‐pan‐cytokeratin , anti‐Melan‐A , anti‐LYVE‐1 , anti‐fibronectin , anti‐CD31 , anti‐caspase‐3 , anti‐mouse/human Ki‐67  antibodies, or anti‐HSP70 . Dye‐conjugated secondary antibodies  were used for the addition of the fluorescence signals. DAPI  was used to stain the cell nuclei. The stained tissue sections were imaged using a fluorescent confocal microscope and an EVOS FL2 auto microscope or FluoView FV‐10i Olympus Laser Point Scanning Confocal Microscope . After splitting each color channel, the fluorescent area for each color channel was measured with ImageJ . The expression level for each marker was calculated on the basis of normalization with the DAPI signal.4.18Major organs were immediately fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5‐μm‐thick sections, which were then stained with H&E for histological examination. Organ histology was viewed and imaged under light microscopy (EVOS FL2).4.19iv injection of CLCs  was measured by the Infinity Urea kit (Thermo Fisher Scientific) and compared against a standard BUN solution of 100 mg/dl (Sigma‐Aldrich) using a VersaMax microplate reader , as per the protocol provided by Thermo Fisher Scientific. The creatinine (Cr) of the serum following iv injection of CLCs  was measured using the microplate reader, according to the instructions provided in the Mouse Creatinine Assay Kit .BUN of the C57BL/6 mice serum following 4.20n) in the experiments was as follows: six for tumor growth analysis, six for lung metastatic foci assay, three for the fluorescent biodistribution, and three for the tissue immunostaining study. The fluorescence in vitro assay was independently conducted three times for statistical analysis. All data are expressed as the mean ± SD from at least three independent samples or experiments. Differences between the two groups were analyzed by an unpaired Student's t‐test. Comparisons between multiple groups were determined using one or two‐way analysis of variance with Holm‐Sidak's post hoc test. A p value <0.05 was considered statistically significant .All statistical analysis was conducted using GraphPad Prism 7 software . The expression level of each marker in the immunostaining experiments was determined by dividing each DAPI level. The sample size ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); validation (lead); visualization (lead). Liwei Jiang: Conceptualization ; data curation (lead); formal analysis (lead); investigation ; methodology . Jing Zhao: Data curation (supporting); investigation ; methodology . Leonard D. Shultz: Funding acquisition ; resources ; supervision . Dale L. Greiner: Methodology ; resources ; supervision . Munhyung Bae: Data curation (supporting); methodology (supporting). Xiaofei Li: Data curation (supporting); methodology (supporting). Farideh Ordikhani: Conceptualization (supporting); formal analysis (supporting); methodology (supporting). Rui Kuai: Supervision (supporting); validation (supporting). John Joseph: Data curation (supporting); formal analysis (supporting); methodology (supporting). Vivek Kasinath: Data curation (supporting); formal analysis (supporting); funding acquisition ; supervision (supporting); validation (supporting). David R. Elmaleh: Conceptualization ; formal analysis (supporting); methodology (supporting); resources (supporting); supervision (supporting). Reza Abdi: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); software (lead); supervision (lead); validation (lead); visualization (lead).All authors declare no conflict of interests.https://publons.com/publon/10.1002/btm2.10273.The peer review history for this article is available at Appendix S1: Supporting informationClick here for additional data file.